Consensus prostate antigens, nucleic acid molecule encoding the same and vaccine and uses comprising the same

ABSTRACT

Provided herein are consensus amino acid sequences of prostate antigens that are capable of breaking tolerance in a targeted species, including PSA, PSMA, STEAP and PSCA antigens. Also provided are nucleic acid sequences that encode one or more consensus amino acid sequences of prostate antigens PSA, PSMA, STEAP and PSCA, as well genetic constructs/vectors and vaccines expressing the sequences. Also provided herein are methods for generating an autoimmune response against prostate cancer cells by administering one or more of the vaccines, proteins, and/or nucleic acid sequences that are provided.

FIELD OF THE INVENTION

The present invention relates to nucleic acid sequences encodingconsensus prostate proteins and fragments thereof; to improved prostatecancer vaccines, improved methods for inducing immune responses againstprostate cancer cells, improved methods for prophylactically and/ortherapeutically immunizing individuals against prostate cancer.

BACKGROUND OF THE INVENTION

Prostate cancer is an important therapeutic immune target. Thedevelopment of an immune therapeutic approach is complex, in thatimmunogens need to be developed that are capable of inducing strongimmune responses including preferably CTL responses.

The direct administration of nucleic acid sequences to vaccinate againstanimal and human diseases has been studied and much effort has focusedon effective and efficient means of nucleic acid delivery in order toyield necessary expression of the desired antigens, resultingimmunogenic response and ultimately the success of this technique.

DNA vaccines have many conceptual advantages over more traditionalvaccination methods, such as live attenuated viruses and recombinantprotein-based vaccines. DNA vaccines are safe, stable, easily produced,and well tolerated in humans with preclinical trials indicating littleevidence of plasmid integration [Martin, T., et al., Plasmid DNA malariavaccine: the potential for genomic integration after intramuscularinjection. Hum Gene Ther, 1999. 10(5): p. 759-68; Nichols, W. W., etal., Potential DNA vaccine integration into host cell genome. Ann N YAcad Sci, 1995. 772: p. 30-9]. In addition, DNA vaccines are well suitedfor repeated administration due to the fact that efficacy of the vaccineis not influenced by pre-existing antibody titers to the vector[Chattergoon, M., J. Boyer, and D. B. Weiner, Genetic immunization: anew era in vaccines and immune therapeutics. FASEB J, 1997. 11(10): p.753-63]. However, one major obstacle for the clinical adoption of DNAvaccines has been a decrease in the platform's immunogenicity whenmoving to larger animals [Liu, M. A. and J. B. Ulmer, Human clinicaltrials of plasmid DNA vaccines. Adv Genet, 2005. 55: p. 25-40]. Recenttechnological advances in the engineering of DNA vaccine immunogen, suchhas codon optimization, RNA optimization and the addition ofimmunoglobulin leader sequences have improved expression andimmunogenicity of DNA vaccines [Andre, S., et al., Increased immuneresponse elicited by DNA vaccination with a synthetic gp120 sequencewith optimized codon usage. J Virol, 1998. 72(2): p. 1497-503; Deml, L.,et al., Multiple effects of codon usage optimization on expression andimmunogenicity of DNA candidate vaccines encoding the humanimmunodeficiency virus type 1 Gag protein. J Virol, 2001. 75(22): p.10991-1001; Laddy, D. J., et al., Immunogenicity of novelconsensus-based DNA vaccines against avian influenza. Vaccine, 2007.25(16): p. 2984-9; Frelin, L., et al., Codon optimization and mRNAamplification effectively enhances the immunogenicity of the hepatitis Cvirus nonstructural 3/4A gene. Gene Ther, 2004. 11(6): p. 522-33].

Recent technological advances in plasmid delivery systems have improvedexpression and immunogenicity of DNA vaccines including technologiessuch as electroporation [Hirao, L. A., et al., Intradermal/subcutaneousimmunization by electroporation improves plasmid vaccine delivery andpotency in pigs and rhesus macaques. Vaccine, 2008. 26(3): p. 440-8;Luckay, A., et al., Effect of plasmid DNA vaccine design and in vivoelectroporation on the resulting vaccine-specific immune responses inrhesus macaques. J Virol, 2007. 81(10): p. 5257-69; Ahlen, G., et al.,In vivo electroporation enhances the immunogenicity of hepatitis C virusnonstructural 3/4A DNA by increased local DNA uptake, proteinexpression, inflammation, and infiltration of CD3+ T cells. J Immunol,2007. 179(7): p. 4741-53].

In addition, studies have suggested that the use of consensus immunogenscan be able to increase the breadth of the cellular immune response ascompared to native antigens alone [Yan, J., et al., Enhanced cellularimmune responses elicited by an engineered HIV-1 subtype Bconsensus-based envelope DNA vaccine. Mol Ther, 2007. 15(2): p. 411-21;Rolland, M., et al., Reconstruction and function of ancestralcenter-of-tree human immunodeficiency virus type 1 proteins. J Virol,2007. 81(16): p. 8507-14]. However, it is recognized that breakingimmune tolerance for cancer antigens and generating autoimmunity is amajor obstacle for cancer vaccines.

There still remains a need for nucleic acid constructs that encodeprostate cancer antigens and for compositions useful to induce immuneresponses against prostate cancer antigens and thus break immunetolerance. There remains a need for effective prophylactic andtherapeutic vaccines against prostate cancer that are economical andeffective.

SUMMARY OF THE PREFERRED EMBODIMENTS

Aspects of the present invention include nucleic acid moleculescomprising a coding sequence encoding one or more proteins selected fromthe group comprising: a) SEQ ID NO:2; a protein that is 98% homologousto SEQ ID NO:2, provided amino acids 69, 78, 80, 82, 102, 110, 137, 139,165, 189, 203, 220, 232 and 248 of SEQ ID NO:2 are conserved; or animmunogenic fragment of SEQ ID NO:2 comprising amino acids correspondingto at least 256 amino acid residues of SEQ ID NO:2, provided amino acids69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 ofSEQ ID NO:2 are conserved; b) SEQ ID NO:4; a protein that is 98%homologous to SEQ ID NO:4, provided amino acids 21, 86, 127, 129, 154,156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 and 275 of SEQ IDNO:4 are conserved; or an immunogenic fragment of SEQ ID NO:4 comprisingamino acids corresponding to at least 274 amino acid residues of SEQ IDNO:4, provided amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206,218, 220, 237, 249, 255, 265, 271 and 275 of SEQ ID NO:4 are conserved;c) SEQ ID NO:6; a protein that is 98% homologous to SEQ ID NO:6,provided amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350,475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO:6 areconserved; or an immunogenic fragment of SEQ ID NO:6 comprising aminoacids corresponding to at least 735 amino acid residues of SEQ ID NO:6,provided amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350,475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO:6 areconserved; d) SEQ ID NO:8; a protein that is 98% homologous to SEQ IDNO:8, provided amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239,336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 ofSEQ ID NO:8 are conserved; or an immunogenic fragment of SEQ ID NO:8comprising amino acids corresponding to at least 751 amino acid residuesof SEQ ID NO:8, provided amino acids 20, 30, 31, 48, 63, 74, 95, 127,173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and750 of SEQ ID NO:8 are conserved; e) SEQ ID NO:10; a protein that is 98%homologous to SEQ ID NO:10; or an immunogenic fragment of SEQ ID NO:10comprising amino acids corresponding to at least 333 amino acid residuesof SEQ ID NO:10; f) SEQ ID NO:12; a protein that is 98% homologous toSEQ ID NO:12; or an immunogenic fragment of SEQ ID NO:12 comprisingamino acids corresponding to at least 349 amino acid residues of SEQ IDNO:12; g) SEQ ID NO:14; a protein that is 98% homologous to SEQ ID NO:14or an immunogenic fragment of SEQ ID NO:14 comprising amino acidscorresponding to at least 129 amino acid residues of SEQ ID NO:14; or h)a signal peptide linked to amino acids 19-131 of SEQ ID NO:14; a proteinthat has a signal peptide linked to an amino acid sequence that is 98%homologous to amino acids 19-131 of SEQ ID NO:14; or protein that has asignal peptide linked to an immunogenic fragment of amino acids 19-131of SEQ ID NO:14, the fragment comprising at least 110 amino acidresidues of SEQ ID NO:14 and linked to a signal peptide. In someembodiments the nucleic acid molecules are chosen from ones encodingproteins a), b), c), or d).

In another aspect, the invention includes methods of treating anindividual who has been diagnosed with prostate cancer comprisingadministering a nucleic acid molecule described herein to an individual.

In another aspect, there are provided proteins selected from the groupconsisting of: a) SEQ ID NO:2; a protein that is 98% homologous to SEQID NO:2, provided amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165,189, 203, 220, 232 and 248 of SEQ ID NO:2 are conserved; or animmunogenic fragment of SEQ ID NO:2 comprising amino acids correspondingto at least 261 amino acid residues of SEQ ID NO:2, provided amino acids69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 ofSEQ ID NO:2 are conserved; b) SEQ ID NO:4; a protein that is 98%homologous to SEQ ID NO:4, provided amino acids 21, 86, 127, 129, 154,156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 and 275 of SEQ IDNO:4 are conserved; or an immunogenic fragment of SEQ ID NO:4 comprisingamino acids corresponding to at least 274 amino acid residues of SEQ IDNO:4, provided amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206,218, 220, 237, 249, 255, 265, 271 and 275 of SEQ ID NO:4 are conserved;c) SEQ ID NO:6; a protein that is 98% homologous to SEQ ID NO:6,provided amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350,475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO:6 areconserved; or an immunogenic fragment of SEQ ID NO:6 comprising aminoacids corresponding to at least 735 amino acid residues of SEQ ID NO:6,provided amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350,475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO:6 areconserved; d) SEQ ID NO:8; a protein that is 98% homologous to SEQ IDNO:8, provided amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239,336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 ofSEQ ID NO:8 are conserved; or an immunogenic fragment of SEQ ID NO:8comprising amino acids corresponding to at least 751 amino acid residuesof SEQ ID NO:8, provided amino acids 20, 30, 31, 48, 63, 74, 95, 127,173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and750 of SEQ ID NO:8 are conserved; e) SEQ ID NO:10; a protein that is 98%homologous to SEQ ID NO:10; or an immunogenic fragment of SEQ ID NO:10comprising amino acids corresponding to at least 333 amino acid residuesof SEQ ID NO:10; f) SEQ ID NO:12; a protein that is 98% homologous toSEQ ID NO:12; or an immunogenic fragment of SEQ ID NO:12 comprisingamino acids corresponding to at least 349 amino acid residues of SEQ IDNO:12; g) SEQ ID NO:14; a protein that is 98% homologous to SEQ IDNO:14; or an immunogenic fragment of SEQ ID NO:14 comprising amino acidscorresponding to at least 129 amino acid residues of SEQ ID NO:14; or h)a signal peptide linked to amino acids 19-131 of SEQ ID NO:14; a proteinthat has a signal peptide linked to an amino acid sequence that is 98%homologous to amino acids 19-131 of SEQ ID NO:14; or protein that has asignal peptide linked to an immunogenic fragment of amino acids 19-131of SEQ ID NO:14, the fragment comprising at least 110 amino acidresidues of SEQ ID NO:14 and linked to a signal peptide. In someembodiments, the protein is selected from the group comprising: proteinsa), b), c), or d).

Some aspects of the invention include methods of treating an individualwho has been diagnosed with prostate cancer comprising delivering tosaid individual a protein described herein.

Other aspects of the invention are pharmaceutical compositionscomprising the nucleic acid molecules provided herein and apharmaceutically acceptable excipient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows results from in vitro translation performed to confirm theexpression of the PSA and PSMA antigens.

FIG. 2A shows cellular immunogenicity data. Cellular immunogenicity ofPSA antigens was determined by Interferon-gamma ELISpot.

FIG. 2B shows cellular immunogenicity data. Cellular immunogenicity ofPSA antigens was determined by Interferon-gamma ELISpot.

FIGS. 3A-C shows CD4+ T cell responses as characterized by flowcytometry by displaying graphs showing PSA-specific (left panel),PSMA-specific (middle panel) and total vaccine-specific (right panel)cytokine production: % IFNγ producing CD4+ T cells (FIG. 3A); % IL-2producing CD4+ T cells (FIG. 3B); and % TNFα producing CD4+ T cells(FIG. 3C).

FIGS. 4A-C shows CD8+ T cell responses as characterized by flowcytometry by displaying graphs showing PSA-specific (left panel),PSMA-specific (middle panel) and total vaccine-specific (right panel)cytokine production: % IFNγ producing CD8+ T cells (FIG. 4A); % IL-2producing CD8+ T cells (FIG. 4B); and % TNFα producing CD8+ T cells(FIG. 4C).

FIGS. 5A-B shows ELISA data for PSA-specific antibodies one week afterthe final immunization. (FIG. 5A) PSA IgG endpoint titers. (FIG. 5B)Representative IgG titration curves.

DETAILED DESCRIPTION

Provided herein are consensus sequence prostate proteins and isolatednucleic acid molecules that encode them, and in particular, the prostateantigens prostate specific antigen (PSA), prostate specific membraneantigen (PSMA), six-transmembrane epithelial antigen of the prostateantigen (STEAP) and prostate specific stem cell antigen (PSCA).

The prostate cancer antigens described herein are consensus sequencesderived from a pool of homologous antigens from across multiple species,including the specie that the vaccine is targeted for. The selectedspecies from which antigen sequences are aligned to form a consensusshall be chosen based on close proximity of the species on a phylogenictree, e.g., H. sapiens (humans), M. mulatta (rhesus macaques), and M.fascicularis (cynomolgus monkey). The consensus antigen is not identicalto the native prostate antigen but will have close identity, whichsequences share at least 85%, and preferably 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity. These described consensus cancerantigens are able to break tolerance in the targeted specie (or causeautoimmunity) and generate an effective immune response against theprostate cancer antigen. Provided herein are methods to generate aconsensus cancer antigen based DNA vaccine.

Aspects of the present invention include nucleic acid moleculescomprising a coding sequence encoding one or more proteins selected fromthe group comprising: a) SEQ ID NO:2; a protein that is 98% homologousto SEQ ID NO:2, provided amino acids 69, 78, 80, 82, 102, 110, 137, 139,165, 189, 203, 220, 232 and 248 of SEQ ID NO:2 are conserved; or animmunogenic fragment of SEQ ID NO:2 comprising amino acids correspondingto at least 256 amino acid residues of SEQ ID NO:2, provided amino acids69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 ofSEQ ID NO:2 are conserved; b) SEQ ID NO:4; a protein that is 98%homologous to SEQ ID NO:4, provided amino acids 21, 86, 127, 129, 154,156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 and 275 of SEQ IDNO:4 are conserved; or an immunogenic fragment of SEQ ID NO:4 comprisingamino acids corresponding to at least 274 amino acid residues of SEQ IDNO:4, provided amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206,218, 220, 237, 249, 255, 265, 271 and 275 of SEQ ID NO:4 are conserved;c) SEQ ID NO:6; a protein that is 98% homologous to SEQ ID NO:6,provided amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350,475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO:6 areconserved; or an immunogenic fragment of SEQ ID NO:6 comprising aminoacids corresponding to at least 735 amino acid residues of SEQ ID NO:6,provided amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350,475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO:6 areconserved; d) SEQ ID NO:8; a protein that is 98% homologous to SEQ IDNO:8, provided amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239,336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 ofSEQ ID NO:8 are conserved; or an immunogenic fragment of SEQ ID NO:8comprising amino acids corresponding to at least 751 amino acid residuesof SEQ ID NO:8, provided amino acids 20, 30, 31, 48, 63, 74, 95, 127,173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and750 of SEQ ID NO:8 are conserved; e) SEQ ID NO:10; a protein that is 98%homologous to SEQ ID NO:10; or an immunogenic fragment of SEQ ID NO:10comprising amino acids corresponding to at least 333 amino acid residuesof SEQ ID NO:10; f) SEQ ID NO:12; a protein that is 98% homologous toSEQ ID NO:12; or an immunogenic fragment of SEQ ID NO:12 comprisingamino acids corresponding to at least 349 amino acid residues of SEQ IDNO:12; g) SEQ ID NO:14; a protein that is 98% homologous to SEQ IDNO:14; or an immunogenic fragment of SEQ ID NO:14 comprising amino acidscorresponding to at least 129 amino acid residues of SEQ ID NO:14; or animmunogenic fragment of SEQ ID NO:14 comprising at least 129 amino acidresidues of SEQ ID NO:14; or h) a signal peptide linked to amino acids19-131 of SEQ ID NO:14; a protein that has a signal peptide linked to anamino acid sequence that is 98% homologous to amino acids 19-131 of SEQID NO:14; or protein that has a signal peptide linked to an immunogenicfragment of amino acids 19-131 of SEQ ID NO:14, the fragment comprisingat least 110 amino acid residues of SEQ ID NO:14 and linked to a signalpeptide. Two consensus protein sequences for PSA are disclosed: PSAConsensus Antigen sequence 1 (SEQ ID NO:2) and PSA Consensus Antigensequence 2 (SEQ ID NO:4). Two consensus protein sequences for PSMA aredisclosed: PSMA Consensus Antigen sequence 1 (SEQ ID NO:6) and PSMAConsensus Antigen sequence 2 (SEQ ID NO:8). Two consensus proteinsequences for STEAP (also referred to herein as STEAP1) are disclosed:STEAP Consensus Antigen sequence 1 (SEQ ID NO:10) and STEAP ConsensusAntigen sequence 2 (SEQ ID NO:12). One consensus protein sequence forPSCA is disclosed: PSCA Consensus Antigen sequence (SEQ ID NO:14). SEQID NO:14 includes an IgE signal peptide. In some embodiments, a PSCAConsensus antigen may include amino acids 19-131 of SEQ ID NO:14 linkedto a signal sequence other than the IgE signal in SEQ ID NO:14. In someembodiments the nucleic acid molecules are chosen from ones encodingproteins a), b), c), or d), above. In other embodiments the nucleic acidmolecules are ones encoding one or more proteins selected from the groupcomprising: at least one selected from ones encoding either proteins a)or b), and at least one selected from ones encoding either proteins c)or d).

The nucleic acid molecules can further be molecules encoding one or moreproteins selected from the group comprising: SEQ ID NO:2; SEQ ID NO:4;SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; or SEQ ID NO:14;and preferably, SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; or SEQ ID NO:8.In some embodiments, the nucleic acid molecule of can be ones thatencode one or more proteins selected from the group comprising: at leastone selected from either SEQ ID NO:2 or SEQ ID NO:4, and at least oneselected from either SEQ ID NO:6 or SEQ ID NO:8.

In another aspect, there are provided proteins selected from the groupconsisting of: a) SEQ ID NO:2; a protein that is 98% homologous to SEQID NO:2, provided amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165,189, 203, 220, 232 and 248 of SEQ ID NO:2 are conserved; or animmunogenic fragment of SEQ ID NO:2 comprising amino acids correspondingto at least 256 amino acid residues of SEQ ID NO:2, provided amino acids69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 ofSEQ ID NO:2 are conserved; b) SEQ ID NO:4; a protein that is 98%homologous to SEQ ID NO:4, provided amino acids 21, 86, 127, 129, 154,156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 and 275 of SEQ IDNO:4 are conserved; or an immunogenic fragment of SEQ ID NO:4 comprisingamino acids corresponding to at least 274 amino acid residues of SEQ IDNO:4, provided amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206,218, 220, 237, 249, 255, 265, 271 and 275 of SEQ ID NO:4 are conserved;c) SEQ ID NO:6; a protein that is 98% homologous to SEQ ID NO:6,provided amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350,475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO:6 areconserved; or an immunogenic fragment of SEQ ID NO:6 comprising aminoacids corresponding to at least 735 amino acid residues of SEQ ID NO:6,provided amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350,475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO:6 areconserved; d) SEQ ID NO:8; a protein that is 98% homologous to SEQ IDNO:8, provided amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239,336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 ofSEQ ID NO:8 are conserved; or an immunogenic fragment of SEQ ID NO:8comprising amino acids corresponding to at least 751 amino acid residuesof SEQ ID NO:8, provided amino acids 20, 30, 31, 48, 63, 74, 95, 127,173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and750 of SEQ ID NO:8 are conserved; e) SEQ ID NO:10; a protein that is 98%homologous to SEQ ID NO:10; or an immunogenic fragment of SEQ ID NO:10comprising amino acids corresponding to at least 333 amino acid residuesof SEQ ID NO:10; f) SEQ ID NO:12; a protein that is 98% homologous toSEQ ID NO:12; or an immunogenic fragment of SEQ ID NO:12 comprisingamino acids corresponding to at least 349 amino acid residues of SEQ IDNO:12; g) SEQ ID NO:14; a protein that is 98% homologous to SEQ IDNO:14; or an immunogenic fragment of SEQ ID NO:14 comprising amino acidscorresponding to at least 129 amino acid residues of SEQ ID NO:14; or h)a signal peptide linked to amino acids 19-131 of SEQ ID NO:14; a proteinthat has a signal peptide linked to an amino acid sequence that is 98%homologous to amino acids 19-131 of SEQ ID NO:14; or protein that has asignal peptide linked to an immunogenic fragment of amino acids 19-131of SEQ ID NO:14, the fragment comprising at least 110 amino acidresidues of SEQ ID NO:14 and linked to a signal peptide. In someembodiments, the protein is selected from the group comprising: proteinsa), b), c), or d). In other embodiments the proteins are ones encodingone or more proteins selected from the group comprising: at least oneselected from either proteins a) or b), and at least one selected fromeither proteins c) or d).

The proteins can further be proteins selected from the group comprising:SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ IDNO:12; or SEQ ID NO:14; and preferably, SEQ ID NO:2; SEQ ID NO:4; SEQ IDNO:6; or SEQ ID NO:8. In some embodiments, the proteins can be onesselected from the group comprising: at least one selected from eitherSEQ ID NO:2 or SEQ ID NO:4, and at least one selected from either SEQ IDNO:6 or SEQ ID NO:8.

Nucleic acid coding sequences have been generated to improve andoptimize expression. The codons used in these nucleic acid moleculeswere selected to generate RNA having reduced secondary structureformation due to intramolecular hybridization. Nucleic acid sequencesencoding PSA Consensus Antigen sequence 1 (SEQ ID NO:1) and PSAConsensus Antigen sequence 2 (SEQ ID NO:3) are disclosed. Likewise,nucleic acid coding sequence for PSMA Consensus Antigen sequence 1 (SEQID NO:5 of nucleotides 1-2250 of SEQ ID NO:5) and PSMA Consensus Antigensequence 2 (SEQ ID NO:7 or nucleotides 1-2301 of SEQ ID NO:7) as well asSTEAP Consensus Antigen sequence 1 (SEQ ID NO:9), STEAP ConsensusAntigen sequence 2 (SEQ ID NO:11) and PSCA Consensus Antigen sequence(SEQ ID NO:13) are provided. Also provides are nucleic acid sequencesthat are 98% homologous to SEQ ID NO:1 and encode either PSA ConsensusAntigen sequence 1 (SEQ ID NO:2) or a protein up to 98% homologous toSEQ ID NO:2, preferably including amino acids 69, 78, 80, 82, 102, 110,137, 139, 165, 189, 203, 220, 232 and 248 of SEQ ID NO:2, and nucleicacid sequences that are 98% homologous to SEQ ID NO:3 and encode eitherPSA Consensus Antigen sequence 2 (SEQ ID NO:4) or a protein up to 98%homologous to SEQ ID NO:4, preferably including amino acids 21, 86, 127,129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 and 275of SEQ ID NO:4. Likewise, nucleic acid sequences that are 98% homologousto nucleotides 2250 of SEQ ID NO:5 and encode either PSMA ConsensusAntigen sequence 1 (SEQ ID NO:6) or a protein up to 98% homologous toSEQ ID NO:6, preferably including amino acids 14, 15, 32, 47, 58, 79,111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and734 of SEQ ID NO:6, or nucleic acid sequences that are 98% homologous tonucleotides 2301 of SEQ ID NO:7 and encode either PSMA Consensus Antigensequence 1 (SEQ ID NO:8) or a protein up to 98% homologous to SEQ IDNO:8, preferably including amino acids 20, 30, 31, 48, 63, 74, 95, 127,173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and750 of SEQ ID NO:8 as well as nucleotides 98% homologous to SEQ ID NO 9and encode either STEAP Consensus Antigen sequence 1 (SEQ ID NO:10) or aprotein that is up to 98% homologous to SEQ ID NO:10, nucleotides 98%homologous to SEQ ID NO:11 and encode either STEAP Consensus Antigensequence 2 (SEQ ID NO:12) or a protein that is up to 98% homologous toSEQ ID NO:12, and nucleotides 98% homologous to SEQ ID NO:13 and encodeswith PSCA Consensus Antigen sequence (SEQ ID NO:14) or a protein that isup to 98% homologous to SEQ ID NO:14. In some embodiments nucleic acidmolecules encode a protein that comprises an IgE signal peptide (forexample, SEQ ID NO:3 which encodes SEQ ID NO:4; nucleotides 1-2301 ofSEQ ID NO:7 which encodes SEQ ID NO:8; SEQ ID NO:11 which encodes SEQ IDNO:12, and SEQ ID NO:13 which encodes SEQ ID NO:14).

Compositions comprising nucleic acid molecules which comprise the codingsequences of the isolated nucleic acid molecules provided herein may beuseful for inducing immune responses against a prostate protein whenadministered into an animal. Compositions containing one or more ofthese nucleic acid sequences may be used as vaccines or vaccinecomponents to prophylactically or therapeutically immunize againstprostate cancer. Likewise, compositions comprising consensus proteinsmay be useful for inducing immune responses against a prostate proteinwhen administered into an animal. Combinations of compositionscomprising nucleic acid molecules which comprise the coding sequences ofthe isolated nucleic acid molecules provided herein may be useful toinduce immune responses against a prostate protein and may collectivelybe used as vaccines or vaccine components to prophylactically ortherapeutically immunize against prostate cancer. Likewise, compositionscomprising consensus proteins may be useful for inducing immuneresponses against a prostate protein when administered into an animal.Compositions containing one or more of these consensus proteins may beused as vaccines or vaccine components to prophylactically ortherapeutically immunize against prostate cancer.

Vaccines are provided which comprises nucleic acid sequences providedherein. In some embodiments, vaccines are provided which comprisesnucleic acid sequences encoding one or more consensus prostate antigensselected from the group consisting of: consensus PSA antigen 1,consensus PSA antigen 2, consensus PSMA antigen 1, consensus PSMAantigen 2, consensus STEAP antigen 1, consensus STEAP antigen 2, andconsensus PSCA. Methods of inducing immune responses using nucleic acidsequences encoding one or more prostate antigens selected from the groupconsisting of: consensus PSA antigen 1, consensus PSA antigen 2,consensus PSMA antigen 1, consensus PSMA antigen 2, consensus STEAPantigen 1, consensus STEAP antigen 2, and consensus PSCA.

Vaccines which comprise one or more of consensus PSA antigen 1,consensus PSA antigen 2, consensus PSMA antigen 1, consensus PSMAantigen 2, consensus STEAP antigen 1, consensus STEAP antigen 2, andconsensus PSCA are provided. Methods of inducing immune responses usingone or more of consensus PSA antigen 1, consensus PSA antigen 2,consensus PSMA antigen 1, consensus PSMA antigen 2, consensus STEAPantigen 1, consensus STEAP antigen 2, and consensus PSCA are alsoprovided.

Methods of protecting an individual against prostate cancer or oftreating an individual who has been identified as having prostate cancerare provided. The methods comprise the step of: administering to saidindividual an effective amount of one or more nucleic acid moleculescomprising one or more nucleic acid sequences provided herein. In somemethods, the delivery of the nucleic acid molecules is facilitated byelectroporation of the targeted tissue or the tissue that receives thenucleic acid molecules. The nucleic acid sequence is expressed in cellsof the individual and an immune response is induced against the prostateprotein encoded by the nucleic acid sequence.

1. DEFINITIONS

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6, 9, and 7.0 are explicitlycontemplated.

a. Adjuvant

“Adjuvant” as used herein means any molecule added to the DNA plasmidvaccines described herein to enhance the immunogenicity of the antigensencoded by the DNA plasmids and the encoding nucleic acid sequencesdescribed hereinafter.

b. Antibody “Antibody” as used herein means an antibody of classes IgG,IgM, IgA, IgD or IgE, or fragments, fragments or derivatives thereof,including Fab, F(ab′)2, Fd, and single chain antibodies, diabodies,bispecific antibodies, bifunctional antibodies and derivatives thereof.The antibody can be an antibody isolated from the serum sample ofmammal, a polyclonal antibody, affinity purified antibody, or mixturesthereof which exhibits sufficient binding specificity to a desiredepitope or a sequence derived therefrom.

c. Coding Sequence

“Coding sequence” or “encoding nucleic acid” as used herein means thenucleic acids (RNA or DNA molecule) that comprise a nucleotide sequencewhich encodes a protein. The coding sequence can further includeinitiation and termination signals operably linked to regulatoryelements including a promoter and polyadenylation signal capable ofdirecting expression in the cells of an individual or mammal to whom thenucleic acid is administered.

d. Complement

“Complement” or “complementary” as used herein means a nucleic acid canmean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairingbetween nucleotides or nucleotide analogs of nucleic acid molecules.

e. Consensus or Consensus Sequence

“Consensus” or “consensus sequence” as used herein means a polypeptidesequence based on analysis of an alignment of multiple subtypes of aparticular prostate antigen.

Nucleic acid sequences that encode a consensus polypeptide sequence maybe prepared. Vaccines comprising proteins that comprise consensussequences and/or nucleic acid molecules that encode such proteins can beused to induce broad immunity against a particular prostate antigen.

f. Electroporation

“Electroporation,” “electro-permeabilization,” or “electro-kineticenhancement” (“EP”) as used interchangeably herein means the use of atransmembrane electric field pulse to induce microscopic pathways(pores) in a bio-membrane; their presence allows biomolecules such asplasmids, oligonucleotides, siRNA, drugs, ions, and water to pass fromone side of the cellular membrane to the other.

g. Fragment

“Fragment” as used herein with respect to nucleic acid sequences means anucleic acid sequence or a portion thereof, that encodes a polypeptidecapable of eliciting an immune response in a mammal that cross reactswith a full length prostate antigen. The fragments can be DNA fragmentsselected from at least one of the various nucleotide sequences thatencode the consensus amino acid sequences and constructs comprising suchsequences. DNA fragments can comprise coding sequences for theimmunoglobulin leader such as IgE or IgG sequences. DNA fragments canencode the protein fragments set forth below.

“Fragment” with respect to polypeptide sequences means a polypeptidecapable of eliciting an immune response in a mammal that cross reactswith a prostate antigen, including, e.g. PSA, PSMA, STEAP and PSCA.

The human PSA sequence is about 261 amino acids. Fragments of PSAconsensus antigen 1 may comprise at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% of SEQ ID NO:2, and preferably 98% or 99%,provided the fragments include one or more of amino acids 69, 78, 80,82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248. Fragments ofPSA consensus antigen 1 may comprise 255, 256, 257, 258, 259 or 260amino acids of SEQ ID NO:2, but preferably 256 amino acids or more.Fragments of PSA consensus antigen 2 may comprise at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:4, and preferably98% or 99%, provided the fragments include one or more of amino acids21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265,271 and 275. All such fragments of PSA consensus antigen 2 may alsooptionally exclude amino acids 1-17. In some embodiments, fragments ofPSA consensus antigen 2 may optionally comprise one or more of aminoacids 1-17 and of the amino acids from amino acid 18 to amino acid 278,fragments of PSA consensus antigen 2 may also comprise 255, 256, 257,258, 259 or 260 amino acids of SEQ ID NO:4, but preferably 274 aminoacids or more.

The human PSMA sequence is about 749-750 amino acids. Fragments of PSMAconsensus antigen 1 may comprise at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% of SEQ ID NO:6, and preferably 98% or 99%,provided the fragments include one or more of amino acids 14, 15, 32,47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660,663, 733 and 734. Fragments of PSMA consensus antigen 1 may comprise745, 746, 747, 748 or 749 amino acids of SEQ ID NO:6, but preferably 735amino acids or more. Fragments of PSMA consensus antigen 2 may compriseat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ IDNO:8, and preferably 98% or 99%, provided the fragments include one ormore of amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366,491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750. All suchfragments of PSA consensus antigen 2 may also optionally exclude aminoacids 1-16. In some embodiments, fragments of PSA consensus antigen 2may optionally comprise one or more of amino acids 1-16 and of the aminoacids from amino acid 17 to amino acid 766, fragments of PSMA consensusantigen 2 may also comprise 760, 761, 762, 763, 764 or 765 amino acidsof SEQ ID NO:8, but preferably 751 amino acids or more.

The human STEAP sequence is about 339 amino acids. Consensus STEAPsequences may comprise amino acid sequences for the immunoglobulinleader such as IgE or IgG. Consensus STEAP antigen 2 contains an 18amino acid leader sequence in place of the methionine at position 1.Fragments of PSMA consensus antigen 2 may comprise a leader sequence andat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of aminoacids 18-356 of SEQ ID NO:12, and preferably 98% or 99%. Fragments ofPSMA consensus antigen 1 may comprise amino acids 1-350, 1-351, 1-352,1-353, 1-354 or 1-355 of SEQ ID NO:12.

The human PSCA sequence is about 114 amino acids. Consensus STEAPsequences may comprise amino acid sequences for the immunoglobulinleader such as IgE or IgG. Consensus PSCA antigen contains an 18 aminoacid leader sequence in place of the methionine at position 1. Fragmentsof PSCA consensus antigen may comprise a leader sequence and at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of amino acids18-131 of SEQ ID NO:14, and preferably 98% or 99%. Fragments of PSMAconsensus antigen 1 may comprise amino acids 1-125, 1-126, 1-127, 1-128,1-129 or 1-130 of SEQ ID NO:14.

h. Genetic Construct

As used herein, the term “genetic construct” refers to the DNA or RNAmolecules that comprise a nucleotide sequence which encodes a protein.The coding sequence includes initiation and termination signals operablylinked to regulatory elements including a promoter and polyadenylationsignal capable of directing expression in the cells of the individual towhom the nucleic acid molecule is administered. As used herein, the term“expressible form” refers to gene constructs that contain the necessaryregulatory elements operable linked to a coding sequence that encodes aprotein such that when present in the cell of the individual, the codingsequence will be expressed.

i. Homology

Homology of multiple sequence alignments and phylogram were generatedusing ClustalW, a general purpose multiple sequence alignment programfor DNA or proteins.

j. Identical

“Identical” or “identity” as used herein in the context of two or morenucleic acids or polypeptide sequences, means that the sequences have aspecified percentage of residues that are the same over a specifiedregion. The percentage can be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) can be considered equivalent.Identity can be performed manually or by using a computer sequencealgorithm such as BLAST or BLAST 2.0.

k. Immune Response

“Immune response” as used herein means the activation of a host's immunesystem, e.g., that of a mammal, in response to the introduction ofantigen such as a prostate consensus antigen. The immune response can bein the form of a cellular or humoral response, or both.

l. Nucleic Acid

“Nucleic acid” or “oligonucleotide” or “polynucleotide” as used hereinmeans at least two nucleotides covalently linked together. The depictionof a single strand also defines the sequence of the complementarystrand. Thus, a nucleic acid also encompasses the complementary strandof a depicted single strand. Many variants of a nucleic acid can be usedfor the same purpose as a given nucleic acid. Thus, a nucleic acid alsoencompasses substantially identical nucleic acids and complementsthereof. A single strand provides a probe that can hybridize to a targetsequence under stringent hybridization conditions. Thus, a nucleic acidalso encompasses a probe that hybridizes under stringent hybridizationconditions.

Nucleic acids can be single stranded or double stranded, or can containportions of both double stranded and single stranded sequence. Thenucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid can contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids can be obtained by chemical synthesismethods or by recombinant methods.

m. Operably Linked

“Operably linked” as used herein means that expression of a gene isunder the control of a promoter with which it is spatially connected. Apromoter can be positioned 5′ (upstream) or 3′ (downstream) of a geneunder its control. The distance between the promoter and a gene can beapproximately the same as the distance between that promoter and thegene it controls in the gene from which the promoter is derived. As isknown in the art, variation in this distance can be accommodated withoutloss of promoter function.

n. Promoter

“Promoter” as used herein means a synthetic or naturally-derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter can comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of same. A promoter can also comprise distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A promoter can bederived from sources including viral, bacterial, fungal, plants,insects, and animals. A promoter can regulate the expression of a genecomponent constitutively, or differentially with respect to cell, thetissue or organ in which expression occurs or, with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents. Representative examples of promoters include thebacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lacoperator-promoter, tac promoter, SV40 late promoter, SV40 earlypromoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40late promoter and the CMV IE promoter.

o. Stringent Hybridization Conditions

“Stringent hybridization conditions” as used herein means conditionsunder which a first nucleic acid sequence (e.g., probe) will hybridizeto a second nucleic acid sequence (e.g., target), such as in a complexmixture of nucleic acids. Stringent conditions are sequence-dependentand will be different in different circumstances. Stringent conditionscan be selected to be about 5-10° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength pH.The T_(m) can be the temperature (under defined ionic strength, pH, andnucleic concentration) at which 50% of the probes complementary to thetarget hybridize to the target sequence at equilibrium (as the targetsequences are present in excess, at T_(m), 50% of the probes areoccupied at equilibrium). Stringent conditions can be those in which thesalt concentration is less than about 1.0 M sodium ion, such as about0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g.,about 10-50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions can alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal can be atleast 2 to 10 times background hybridization. Exemplary stringenthybridization conditions include the following: 50% formamide, 5×SSC,and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65°C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

p. Substantially Complementary

“Substantially complementary” as used herein means that a first sequenceis at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%identical to the complement of a second sequence over a region of 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450,540, 630, 720, 810, 900, 990, 1080, 1170, 1260, 1350, 1440, 1530, 1620,1710, 1800, 1890, 1980, 2070 or more nucleotides or amino acids, or thatthe two sequences hybridize under stringent hybridization conditions.

q. Substantially Identical

“Substantially identical” as used herein means that a first and secondsequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 180, 270, 360, 450, 540, 630, 720, 810, 900, 990,1080, 1170, 1260, 1350, 1440, 1530, 1620, 1710, 1800, 1890, 1980, 2070or more nucleotides or amino acids, or with respect to nucleic acids, ifthe first sequence is substantially complementary to the complement ofthe second sequence.

r. Subtype or Serotype

“Subtype” or “serotype”: as used herein, interchangeably, and inreference to prostate cancer antigens, means genetic variants of aprostate cancer antigen such that one subtype (or variant) is recognizedby an immune system apart from a different subtype.

s. Variant

“Variant” used herein with respect to a nucleic acid means (i) a portionor fragment of a referenced nucleotide sequence; (ii) the complement ofa referenced nucleotide sequence or portion thereof; (iii) a nucleicacid that is substantially identical to a referenced nucleic acid or thecomplement thereof; or (iv) a nucleic acid that hybridizes understringent conditions to the referenced nucleic acid, complement thereof,or a sequences substantially identical thereto.

“Variant” with respect to a peptide or polypeptide that differs in aminoacid sequence by the insertion, deletion, or conservative substitutionof amino acids, but retain at least one biological activity. Variant canalso mean a protein with an amino acid sequence that is substantiallyidentical to a referenced protein with an amino acid sequence thatretains at least one biological activity. A conservative substitution ofan amino acid, i.e., replacing an amino acid with a different amino acidof similar properties (e.g., hydrophilicity, degree and distribution ofcharged regions) is recognized in the art as typically involving a minorchange. These minor changes can be identified, in part, by consideringthe hydropathic index of amino acids, as understood in the art. Kyte etal., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an aminoacid is based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retains protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids can also be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101,incorporated fully herein by reference. Substitution of amino acidshaving similar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. Substitutions can be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

t. Vector

“Vector” as used herein means a nucleic acid sequence containing anorigin of replication. A vector can be a vector, bacteriophage,bacterial artificial chromosome or yeast artificial chromosome. A vectorcan be a DNA or RNA vector. A vector can be a self-replicatingextrachromosomal vector, and preferably, is a DNA plasmid.

2. CONSENSUS PROSTATE ANTIGENS

Provided herein are consensus antigens capable of eliciting an immuneresponse in a mammal against a prostate antigen. The consensus antigencan comprise epitopes that make them particularly effective asimmunogens against prostate cancer cells can be induced. The consensusprostate antigen can comprise the full length translation product, avariant thereof, a fragment thereof or a combination thereof.

Seven different consensus prostate antigens have been designed. Two ofthe consensus prostate antigens are consensus PSA antigen 1 (SEQ IDNO:2) and consensus PSA antigen 2 (SEQ ID NO:4). Two of the consensusprostate antigens are consensus PSMA antigen 1 (SEQ ID NO:6) andconsensus PSMA antigen 2 (SEQ ID NO:8). Two of the consensus prostateantigens are consensus STEAP antigen 1 (SEQ ID NO:10) and consensusSTEAP antigen 2 (SEQ ID NO:12). One of the consensus prostate antigensis consensus PSCA antigen (SEQ ID NO:14). Proteins may comprisesequences homologous to the prostate antigens, fragments of the prostateantigens and proteins with sequences homologous to fragments of theprostate antigens.

Consensus PSA antigen 1 (SEQ ID NO:2) is about 91% homologous to humanPSA sequences, about 95% homologous to M. fascicuaris PSA and about 96%homologous to M. mulatta PSA. Consensus PSA antigen 1 differs from humanPSA sequences at amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165,189, 203, 220, 232 and 248 of SEQ ID NO:2.

Consensus PSA antigen 2 (SEQ ID NO:4) is about 90-91% homologous tohuman PSA sequences, about 95% homologous to M. fascicuaris PSA andabout 95% homologous to M. mulatta PSA. Consensus PSA antigen 2comprises a leader sequence at its N terminus Consensus PSA antigen 2also differs from human PSA sequences at amino acids 21, 86, 127, 129,154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 and 275 ofSEQ ID NO:4.

Consensus PSMA antigen 1 (SEQ ID NO:6) is about 96% homologous to humanPSMA sequences and about 94% homologous to M. mulatta PSMA. ConsensusPSMA antigen 1 differs from human PSMA sequences at amino acids 14, 15,32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653,660, 663, 733 and 734 of SEQ ID NO:6.

Consensus PSMA antigen 2 (SEQ ID NO:8) is about 96% homologous to humanPSA sequences and about 94% homologous to M. mulatta PSA. Consensus PSMAantigen 2 comprises a leader sequence at its N terminus Consensus PSMAantigen 2 also differs from human PSA sequences at amino acids 20, 30,31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629,640, 669, 676, 679, 749 and 750 of SEQ ID NO:8.

Consensus STEAP antigen 1 (SEQ ID NO:10) is about 94% homologous to somehuman STEAP sequences and about 99% homologous to other human STEAPsequences. Consensus STEAP antigen 1 (SEQ ID NO:10) is also about 94%homologous to M. mulatta PSMA.

Consensus STEAP antigen 2 (SEQ ID NO:12) is about 88% homologous to somehuman STEAP sequences and about 94% homologous to other human STEAPsequences. Consensus STEAP antigen 2 (SEQ ID NO:12) is also about 94%homologous to M. mulatta PSMA. Consensus STEAP antigen 2 comprises aleader sequence at its N terminus.

Consensus PSCA antigen (SEQ ID NO:14) is about 87% homologous to humanPSCA. Consensus PSCA antigen (SEQ ID NO:14) differs from human PSCA byinclusion of a leader sequence at its N terminus.

Proteins may have sequences 98% homologous to PSA Consensus Antigensequence 1 (SEQ ID NO:2), PSA Consensus Antigen sequence 2 (SEQ IDNO:4), PSMA Consensus Antigen sequence 1 (SEQ ID NO:6), PSMA ConsensusAntigen sequence 2 (SEQ ID NO:8), STEAP Consensus Antigen sequence 1(SEQ ID NO:10), STEAP Consensus Antigen sequence 2 (SEQ ID NO:12) orPSCA Consensus Antigen sequence (SEQ ID NO:14).

Proteins may have sequences 99% homologous to PSA Consensus Antigensequence 1 (SEQ ID NO:2), PSA Consensus Antigen sequence 2 (SEQ IDNO:4), PSMA Consensus Antigen sequence 1 (SEQ ID NO:6), PSMA ConsensusAntigen sequence 2 (SEQ ID NO:8), STEAP Consensus Antigen sequence 1(SEQ ID NO:10), STEAP Consensus Antigen sequence 2 (SEQ ID NO:12) orPSCA Consensus Antigen sequence (SEQ ID NO:14).

As noted above, some embodiments comprise a leader sequence at the Nterminus. In some embodiments, the leader sequence is an IgE leadersequence that is SEQ ID NO:16. In some embodiments of the proteinsequences provided herein, SEQ ID NO:16 is removed therefrom. Likewise,in some embodiments of the nucleic acid sequences provided herein, SEQID NO:15 (which encodes SEQ ID NO:16) is removed therefrom.

Accordingly, some embodiments related protein that comprise a signalpeptide linked to SEQ ID NO:2, SEQ ID NO:6, or SEQ ID NO:10 in place ofthe N terminal methionine set forth in the claim (the coding sequence ofthe signal peptide typically includes a stsart codon encoding an Nterminal methionine). Some embodiments relate to a protein thatcomprises a signal peptide linked to amino acid 19-131 of SEQ ID NO:14.Some embodiments related to proteins that comprise a signal peptidelinked to a protein 98% homologous to SEQ ID NO:2 provided amino acids69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 ofSEQ ID NO:2 are conserved. Some embodiments related to proteins thatcomprise a signal peptide linked to a protein 98% homologous to SEQ IDNO:6 provided amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320,350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO:6are conserved. Some embodiments related to proteins that comprise asignal peptide linked to a protein 98% homologous to SEQ ID NO:10, Ineach instance in which the signal peptide is linked at the N terminal itis linked in place of the N terminal methionine set forth in the claim(the coding sequence of the signal peptide typically includes a stsartcodon encoding an N terminal methionine). Some embodiments relate to aprotein that comprises a signal peptide linked to linked to a protein98% homologous to amino acid 19-131 of SEQ ID NO:14. Some embodimentsrelate to a protein that comprises a signal peptide linked to linked toan immunogenic fragment of SEQ ID NO:2 comprising amino acidscorresponding to at least 256 amino acid residues of SEQ ID NO:2,provided amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203,220, 232 and 248 of SEQ ID NO:2 are conserved. Some embodiments relateto a protein that comprises a signal peptide linked to linked to animmunogenic fragment of SEQ ID NO:6 comprising amino acids correspondingto at least 735 amino acid residues of SEQ ID NO:6, provided amino acids14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613,624, 653, 660, 663, 733 and 734 of SEQ ID NO:6 are conserved. Someembodiments relate to a protein that comprises a signal peptide linkedto an immunogenic fragment of SEQ ID NO:10 comprising amino acidscorresponding to at least 333 amino acid residues of SEQ ID NO:10. Someembodiments relate to a protein that comprises a signal peptide linkedto linked to protein that has a signal peptide linked to an immunogenicfragment of amino acids 19-131 of SEQ ID NO:14, the fragment comprisingat least 110 amino acid residues of SEQ ID NO:14.

3. GENETIC SEQUENCES, CONSTRUCTS AND PLASMIDS

Nucleic acid molecules encoding the consensus amino acid sequences weregenerated to optimize stability and expression in humans. Codonselection was determined based upon, inter alia, an effort to minimizeintramolecular interactions and secondary structure formation as well asusing codons which result in improved expression. Vaccines may compriseone or more nucleic acid sequences that encode one or more of theconsensus versions of the immunogenic proteins selected from this groupof sequences generated to optimize stability and expression in humans.Nucleic acid sequences incorporating coding sequence for the IgE leaderat the 5′ end of the optimized, consensus encoding nucleic acid sequencewere generated which encoded proteins having the IgE leader sequence atthe N terminus of the consensus amino acid sequence. In someembodiments, the nucleic acid sequence that encodes the IgE leader isSEQ ID NO:15

Nucleic acid sequences are provided which encode PSA Consensus Antigensequence 1 (protein sequence SEQ ID NO:2; nucleic acid sequence SEQ IDNO:1), PSA Consensus Antigen sequence 2 (protein sequence SEQ ID NO:4;nucleic acid sequence SEQ ID NO:3), PSMA Consensus Antigen sequence 1(protein sequence SEQ ID NO:6; nucleic acid sequence having nucleotides1-2250 of SEQ ID NO:5), PSMA Consensus Antigen sequence 2 (proteinsequence SEQ ID NO:8; nucleic acid sequence having nucleotides 1-2301 ofSEQ ID NO:7), STEAP Consensus Antigen sequence 1 (protein sequence SEQID NO:10; nucleic acid sequence SEQ ID NO:9), STEAP Consensus Antigensequence 2 (protein sequence SEQ ID NO:12; nucleic acid sequence SEQ IDNO:11) or PSCA Consensus Antigen sequence (protein sequence SEQ IDNO:14; nucleic acid sequence SEQ ID NO:13). The nucleic acid sequenceSEQ ID NO:5 which encodes PSMA Consensus Antigen sequence 1 comprises,in addition to PSMA encoding nucleotides, an additional 9 codons (27nucleotides) immediately before the stop codons which encode the HA Tag(SEQ ID NO:32), not shown in SEQ ID NO:6. The HA Tag is peptide sequencethat corresponds to an influenza epitope useful for among other thingsdetection of protein. expression using commercially available anti-HATag antibodies. SEQ ID NO:5 encodes SEQ ID NO:6 plus an additional 9amino acid sequence SEQ ID NO:32 linked to at its N terminus to the Cterminus of SEQ ID NO:6. In some embodiments, the PSMA-1 Consensusantigen is encoded by SEQ ID NO:5 and comprises a proteins having anamino acid sequence of SEQ ID NO:6 linked at its C terminus to the Nterminus of SEQ ID NO:32. In some embodiments, the PSMA-1 Consensusantigen is encoded by nucleotides 1-2250 of SEQ ID NO:5 and comprises aproteins having an amino acid sequence of SEQ ID NO:6. The codingsequence having nucleotides 1-2250 of SEQ ID NO:5 has one or more stopcodons at its 3′ end. The nucleic acid sequence SEQ ID NO:7 whichencodes PSMA Consensus Antigen sequence 2 comprises, in addition tonucleotides encoding the IgE signal linked to the PSMA, protein plus anadditional 9 codons (27 nucleotides) immediately before the stop codonswhich encode the HA Tag (SEQ ID NO:32), not shown in SEQ ID NO:8. SEQ IDNO:7 encodes SEQ ID NO:8 plus an additional 9 amino acid sequence SEQ IDNO:32 linked to at its N terminus to the C terminus of SEQ ID NO:8. Insome embodiments, the PSMA-2 Consensus antigen is encoded by SEQ ID NO:7and comprises a proteins having an amino acid sequence of SEQ ID NO:8linked at its C terminus to the N terminus of SEQ ID NO:32. In someembodiments, the PSMA-2 Consensus antigen is encoded by nucleotides1-2301 of SEQ ID NO:7 and comprises a proteins having an amino acidsequence of SEQ ID NO:8. The coding sequence having nucleotides 1-2301of SEQ ID NO:7 has one or more stop codons at its 3′ end.

Isolated nucleic acid molecules can encode proteins that have sequences98% homologous to PSA Consensus Antigen sequence 1 (SEQ ID NO:2),provided amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203,220, 232 and 248 of SEQ ID NO:2 are conserved, PSA Consensus Antigensequence 2 (SEQ ID NO:4), provided amino acids 21, 86, 127, 129, 154,156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 and 275 of SEQ IDNO:4 are conserved, PSMA Consensus Antigen sequence 1 (SEQ ID NO:6),provided amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350,475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO:6 areconserved, PSMA Consensus Antigen sequence 2 (SEQ ID NO:8), providedamino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491,515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 of SEQ ID NO:8 areconserved, STEAP Consensus Antigen sequence 1 (SEQ ID NO:10), STEAPConsensus Antigen sequence 2 (SEQ ID NO:12) or PSCA Consensus Antigensequence (SEQ ID NO:14).

Isolated nucleic acid molecules can encode proteins that have sequences99% homologous to PSA Consensus Antigen sequence 1 (SEQ ID NO:2),provided amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203,220, 232 and 248 of SEQ ID NO:2 are conserved, PSA Consensus Antigensequence 2 (SEQ ID NO:4), provided amino acids 21, 86, 127, 129, 154,156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 and 275 of SEQ IDNO:4 are conserved, PSMA Consensus Antigen sequence 1 (SEQ ID NO:6),provided amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350,475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO:6 areconserved, PSMA Consensus Antigen sequence 2 (SEQ ID NO:8), providedamino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491,515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 of SEQ ID NO:8 areconserved, STEAP Consensus Antigen sequence 1 (SEQ ID NO:10), STEAPConsensus Antigen sequence 2 (SEQ ID NO:12) or PSCA Consensus Antigensequence (SEQ ID NO:14).

Isolated nucleic acid molecules can encode proteins that have sequences98% homologous to the sequence encoding PSA Consensus Antigen sequence 1(SEQ ID NO:1), PSA Consensus Antigen sequence 2 (SEQ ID NO:3), PSMAConsensus Antigen sequence 1 (SEQ ID NO:5 or preferably nucleotides1-2250 of SEQ ID NO:5), PSMA Consensus Antigen sequence 2 (SEQ ID NO:7or preferably nucleotides 1-2301 of SEQ ID NO:7), STEAP ConsensusAntigen sequence 1 (SEQ ID NO:9), STEAP Consensus Antigen sequence 2(SEQ ID NO:11) or PSCA Consensus Antigen sequence (SEQ ID NO:13).

Isolated nucleic acid molecules can encode proteins that have sequences99% homologous to the sequence encoding PSA Consensus Antigen sequence 1(SEQ ID NO:1), PSA Consensus Antigen sequence 2 (SEQ ID NO:3), PSMAConsensus Antigen sequence 1 (SEQ ID NO:5 or preferably nucleotides1-2250 of SEQ ID NO:5), PSMA Consensus Antigen sequence 2 (SEQ ID NO:70r preferably nucleotides 1-2301 of SEQ ID NO:7), STEAP ConsensusAntigen sequence 1 (SEQ ID NO:9), STEAP Consensus Antigen sequence 2(SEQ ID NO:11) or PSCA Consensus Antigen sequence (SEQ ID NO:13).

Isolated nucleic acid molecules can encode proteins that comprise aleader sequence at the N terminus. In some embodiments, the nucleic acidmolecules can encode the IgE leader sequence that is SEQ ID NO:16. Insome embodiments isolated nucleic acid molecules can encode proteinsthat comprise a signal peptide linked to SEQ ID NO:2, SEQ ID NO:6, orSEQ ID NO:10 in place of the N terminal methionine set forth in theclaim (the coding sequence of the signal peptide typically includes astsart codon encoding an N terminal methionine). In some embodimentsisolated nucleic acid molecules can encode proteins that comprise asignal peptide linked to amino acid 19-131 of SEQ ID NO:14. In someembodiments isolated nucleic acid molecules can encode proteins thatcomprise a signal peptide linked to a protein 98% homologous to SEQ IDNO:2 provided amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189,203, 220, 232 and 248 of SEQ ID NO:2 are conserved. In some embodimentsisolated nucleic acid molecules can encode proteins that comprise asignal peptide linked to a protein 98% homologous to SEQ ID NO:6provided amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350,475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO:6 areconserved. In some embodiments isolated nucleic acid molecules canencode proteins that comprise a signal peptide linked to a protein 98%homologous to SEQ ID NO:10. In instance in which coding sequence for asignal peptide is provides, the signal peptide is linked to the peptidesequence in place of the N terminal methionine set forth in thesequences shown (the coding sequence of the signal peptide typicallyincludes a stsart codon encoding an N terminal methionine). In someembodiments isolated nucleic acid molecules can encode proteins thatcomprise a signal peptide linked to linked to a protein 98% homologousto amino acid 19-131 of SEQ ID NO:14. In some embodiments isolatednucleic acid molecules can encode proteins that comprise a signalpeptide linked to linked to an immunogenic fragment of SEQ ID NO:2comprising amino acids corresponding to at least 256 amino acid residuesof SEQ ID NO:2, provided amino acids 69, 78, 80, 82, 102, 110, 137, 139,165, 189, 203, 220, 232 and 248 of SEQ ID NO:2 are conserved. In someembodiments isolated nucleic acid molecules can encode proteins thatcomprise a signal peptide linked to linked to an immunogenic fragment ofSEQ ID NO:6 comprising amino acids corresponding to at least 735 aminoacid residues of SEQ ID NO:6, provided amino acids 14, 15, 32, 47, 58,79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733and 734 of SEQ ID NO:6 are conserved. S In some embodiments isolatednucleic acid molecules can encode proteins that comprise a signalpeptide linked to an immunogenic fragment of SEQ ID NO:10 comprisingamino acids corresponding to at least 333 amino acid residues of SEQ IDNO:10. In some embodiments isolated nucleic acid molecules can encodeproteins that comprise a signal peptide linked to linked to protein thathas a signal peptide linked to an immunogenic fragment of amino acids19-131 of SEQ ID NO:14, the fragment comprising at least 110 amino acidresidues of SEQ ID NO:14.

Provided herein are genetic constructs that can comprise a nucleic acidsequence that encodes consensus prostate antigen disclosed hereinincluding consensus protein sequences, sequences homologous to consensusprotein sequences, fragments of consensus protein sequences andsequences homologous to fragments of consensus protein sequences. Thegenetic construct can be present in the cell as a functioningextrachromosomal molecule. The genetic construct can be linearminichromosome including centromere, telomers or plasmids or cosmids.

The genetic construct can also be part of a genome of a recombinantviral vector, including recombinant adenovirus, recombinant adenovirusassociated virus and recombinant vaccinia. The genetic construct can bepart of the genetic material in attenuated live microorganisms orrecombinant microbial vectors which live in cells.

The genetic constructs can comprise regulatory elements for geneexpression of the coding sequences of the nucleic acid. The regulatoryelements can be a promoter, an enhancer an initiation codon, a stopcodon, or a polyadenylation signal.

The nucleic acid sequences may make up a genetic construct that can be avector. The vector can be capable of expressing an antigen in the cellof a mammal in a quantity effective to elicit an immune response in themammal. The vector can be recombinant. The vector can compriseheterologous nucleic acid encoding the antigen. The vector can be aplasmid. The vector can be useful for transfecting cells with nucleicacid encoding an antigen, which the transformed host cell is culturedand maintained under conditions wherein expression of the antigen takesplace.

In some embodiments, coding sequences for a single consensus prostateantigen is provided on a single vector. In some embodiments, codingsequences for a multiple consensus prostate antigen are provided on asingle vector. In some embodiments, compositions are provided comprisingcoding sequences for a multiple consensus prostate antigens on multiplevectors, either one antigen per vector or multiple antigens per vector.

In some embodiments, coding sequences for two or more differentconsensus prostate antigens may be provided on a single vector. In someembodiments, the coding sequences may have separate promoterscontrolling expression. In some embodiments, the coding sequences mayhave a single promoters controlling expression with an IRES sequenceseparating coding sequence. The presence of the IRES sequence results inthe separate translation of the transcription product. In someembodiments, the coding sequences may have a single promoterscontrolling expression with coding sequence encoding a proteolyticcleavage peptide sequence separating coding sequences of the antigens. Asingle translation product is produced which is then processed by theprotease that recognizes the protease cleavage site to generate separateprotein molecules. The protease cleave sites used is typicallyrecognized by a protease endogenously present in the cell whereexpression occurs. In some embodiments, a separate coding sequence for aprotease may be included to provide for the production of the proteaseneeded to process the polyprotein translation product. In someembodiment, vectors comprise coding sequences for one, two, three, four,five, six or all seven consensus prostate antigens.

In each and every instance set forth herein, coding sequences may beoptimized for stability and high levels of expression. In someinstances, codons are selected to reduce secondary structure formationof the RNA such as that formed due to intramolecular bonding.

The vector can comprise heterologous nucleic acid encoding an antigenand can further comprise an initiation codon, which can be upstream ofthe antigen coding sequence, and a stop codon, which can be downstreamof the antigen coding sequence. The initiation and termination codon canbe in frame with the antigen coding sequence. The vector can alsocomprise a promoter that is operably linked to the antigen codingsequence. The promoter operably linked to the antigen coding sequencecan be a promoter from simian virus 40 (SV40), a mouse mammary tumorvirus (MMTV) promoter, a human immunodeficiency virus (HIV) promotersuch as the bovine immunodeficiency virus (BIV) long terminal repeat(LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV)promoter, a cytomegalovirus (CMV) promoter such as the CMV immediateearly promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcomavirus (RSV) promoter. The promoter can also be a promoter from a humangene such as human actin, human myosin, human hemoglobin, human musclecreatine, or human metalothionein. The promoter can also be a tissuespecific promoter, such as a muscle or skin specific promoter, naturalor synthetic. Examples of such promoters are described in US patentapplication publication no. US20040175727, the contents of which areincorporated herein in its entirety.

The vector can also comprise a polyadenylation signal, which can bedownstream of the consensus prostate antigen coding sequence. Thepolyadenylation signal can be a SV40 polyadenylation signal, LTRpolyadenylation signal, bovine growth hormone (bGH) polyadenylationsignal, human growth hormone (hGH) polyadenylation signal, or humanβ-globin polyadenylation signal. The SV40 polyadenylation signal can bea polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego,Calif.).

The vector can also comprise an enhancer upstream of the consensusprostate antigen coding sequence. The enhancer can be necessary for DNAexpression. The enhancer can be human actin, human myosin, humanhemoglobin, human muscle creatine or a viral enhancer such as one fromCMV, HA, RSV or EBV. Polynucleotide function enhances are described inU.S. Pat. Nos. 5,593,972, 5,962,428, and WO94/016737, the contents ofeach are fully incorporated by reference.

The vector can also comprise a mammalian origin of replication in orderto maintain the vector extrachromosomally and produce multiple copies ofthe vector in a cell. The vector can be pVAX1, pCEP4 or pREP4 fromInvitrogen (San Diego, Calif.), which can comprise the Epstein Barrvirus origin of replication and nuclear antigen EBNA-1 coding region,which can produce high copy episomal replication without integration.The backbone of the vector can be pAV0242. The vector can be areplication defective adenovirus type 5 (Ad5) vector.

The vector can also comprise a regulatory sequence, which can be wellsuited for gene expression in a mammalian or human cell into which thevector is administered. The consensus prostate antigen coding sequencecan comprise a codon, which can allow more efficient transcription ofthe coding sequence in the host cell.

The vector can be pSE420 (Invitrogen, San Diego, Calif.), which can beused for protein production in Escherichia coli (E. coli). The vectorcan also be pYES2 (Invitrogen, San Diego, Calif.), which can be used forprotein production in Saccharomyces cerevisiae strains of yeast. Thevector can also be of the MAXBAC™ complete baculovirus expression system(Invitrogen, San Diego, Calif.), which can be used for proteinproduction in insect cells. The vector can also be pcDNA I or pcDNA3(Invitrogen, San Diego, Calif.), which may be used for proteinproduction in mammalian cells such as Chinese hamster ovary (CHO) cells.The vector can be expression vectors or systems to produce protein byroutine techniques and readily available starting materials includingSambrook et al., Molecular Cloning and Laboratory Manual, Second Ed.,Cold Spring Harbor (1989), which is incorporated fully by reference.

Vaccines may comprise one or more of the prostate antigens set forthherein and/or vaccines may comprise one or more nucleic acid sequencesthat encode one or more of the consensus prostate antigen selected fromthis group. Vaccines may comprise one or more of the consensus prostateantigens set forth herein in combination with other immunogenic prostateproteins with sequences other than the consensus sequences disclosedherein including native sequences and/or vaccines may comprise one ormore nucleic acid sequences that encode one or more of the consensusprostate antigens selected from this group in combination with nucleicacid molecules that encode other prostate antigens with sequences otherthan the consensus sequences disclosed herein.

While not being bound by scientific theory, a vaccine that can be usedto elicit an immune response (humoral, cellular, or both) broadlyagainst prostate cancer cells may comprise one or more of the followingnucleic acid sequences that encodes one or more proteins selected fromthe group consisting of: consensus, PSA antigen 1, consensus, PSAantigen 2, consensus, PSMA antigen 1, consensus, PSMA antigen 2,consensus STEAP antigen 1, consensus STEAP antigen 2 and consensus PSCAantigen 1. Coding sequences may also include those provided herein thatcomprise homologous sequences, fragments, and homologous sequences offragments.

Some embodiments provide methods of generating immune responses againstprostate cancer cells comprise administering to an individual one ormore compositions which collectively comprise one or more codingsequences or combinations described herein. Some embodiments providemethods of prophylactically vaccinating an individual against prostatecancer comprise administering one or more compositions whichcollectively comprise one or more coding sequences or combinationsdescribed herein. Some embodiments provide methods of therapeuticallyvaccinating an individual has prostate cancer that compriseadministering one or more compositions which collectively comprise oneor more coding sequences or combinations described herein.

4. PHARMACEUTICAL COMPOSITIONS

Provided herein are pharmaceutical compositions according to the presentinvention which comprise about 1 nanogram to about 10 mg of DNA. In someembodiments, pharmaceutical compositions according to the presentinvention comprise from between: 1) at least 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nanograms, or at least1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230,235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300,305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370,375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440,445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610,615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680,685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750,755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820,825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890,895. 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960,965, 970, 975, 980, 985, 990, 995 or 1000 micrograms, or at least 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mgor more; and 2) up to and including 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95 or 100 nanograms, or up to and including1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230,235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300,305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370,375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440,445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610,615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680,685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750,755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820,825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890,895. 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960,965, 970, 975, 980, 985, 990, 995, or 1000 micrograms, or up to andincluding 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,9, 9.5 or 10 mg. In some embodiments, pharmaceutical compositionsaccording to the present invention comprise about 5 nanogram to about 10mg of DNA. In some embodiments, pharmaceutical compositions according tothe present invention comprise about 25 nanogram to about 5 mg of DNA.In some embodiments, the pharmaceutical compositions contain about 50nanograms to about 1 mg of DNA. In some embodiments, the pharmaceuticalcompositions contain about 0.1 to about 500 micrograms of DNA. In someembodiments, the pharmaceutical compositions contain about 1 to about350 micrograms of DNA. In some embodiments, the pharmaceuticalcompositions contain about 5 to about 250 micrograms of DNA. In someembodiments, the pharmaceutical compositions contain about 10 to about200 micrograms of DNA. In some embodiments, the pharmaceuticalcompositions contain about 15 to about 150 micrograms of DNA. In someembodiments, the pharmaceutical compositions contain about 20 to about100 micrograms of DNA. In some embodiments, the pharmaceuticalcompositions contain about 25 to about 75 micrograms of DNA. In someembodiments, the pharmaceutical compositions contain about 30 to about50 micrograms of DNA. In some embodiments, the pharmaceuticalcompositions contain about 35 to about 40 micrograms of DNA. In someembodiments, the pharmaceutical compositions contain about 100 to about200 microgram DNA. In some embodiments, the pharmaceutical compositionscomprise about 10 microgram to about 100 micrograms of DNA. In someembodiments, the pharmaceutical compositions comprise about 20micrograms to about 80 micrograms of DNA. In some embodiments, thepharmaceutical compositions comprise about 25 micrograms to about 60micrograms of DNA. In some embodiments, the pharmaceutical compositionscomprise about 30 nanograms to about 50 micrograms of DNA. In someembodiments, the pharmaceutical compositions comprise about 35 nanogramsto about 45 micrograms of DNA. In some preferred embodiments, thepharmaceutical compositions contain about 0.1 to about 500 micrograms ofDNA. In some preferred embodiments, the pharmaceutical compositionscontain about 1 to about 350 micrograms of DNA. In some preferredembodiments, the pharmaceutical compositions contain about 25 to about250 micrograms of DNA. In some preferred embodiments, the pharmaceuticalcompositions contain about 100 to about 200 microgram DNA.

The pharmaceutical compositions according to the present invention areformulated according to the mode of administration to be used. In caseswhere pharmaceutical compositions are injectable pharmaceuticalcompositions, they are sterile, pyrogen free and particulate free. Anisotonic formulation is preferably used. Generally, additives forisotonicity can include sodium chloride, dextrose, mannitol, sorbitoland lactose. In some cases, isotonic solutions such as phosphatebuffered saline are preferred. Stabilizers include gelatin and albumin.In some embodiments, a vasoconstriction agent is added to theformulation.

Preferably the pharmaceutical composition is a vaccine, and morepreferably a DNA vaccine.

The vaccine may be a DNA vaccine. The DNA vaccine may comprise aplurality of the same or different plasmids comprising nucleic acidcoding sequences for one or more of consensus prostate antigens. The DNAvaccine may comprise one or more nucleic acid sequences that encode oneor more of consensus prostate antigens. When the DNA vaccine comprisescoding sequences of more than one consensus prostate antigens all suchsequences may be present on a single plasmid, or each such sequences maybe present on a different plasmids.

In some embodiments, vaccines may comprise nucleic acid sequences thatencode one or more of consensus prostate antigens in combination withone or more of consensus prostate antigens.

DNA vaccines are disclosed in U.S. Pat. Nos. 5,593,972, 5,739,118,5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, and5,676,594, which are incorporated herein fully by reference. The DNAvaccine can further comprise elements or reagents that inhibit it fromintegrating into the chromosome. The vaccine can be an RNA of theprostate antigen. The RNA vaccine can be introduced into the cell.

The vaccine can be a recombinant vaccine comprising the geneticconstruct or antigen described above. The vaccine can also comprise oneor more consensus prostate antigens in the form of one or more proteinsubunits, or one or more attenuated viral particles comprising one ormore consensus prostate antigens. The attenuated vaccine can beattenuated live vaccines, killed vaccines and vaccines that userecombinant vectors to deliver foreign genes that encode one or moreconsensus prostate antigens, and well as subunit and glycoproteinvaccines. Examples of attenuated live vaccines, those using recombinantvectors to deliver prostate antigens, subunit vaccines and glycoproteinvaccines are described in U.S. Pat. Nos. 4,510,245; 4,797,368;4,722,848; 4,790,987; 4,920,209; 5,017,487; 5,077,044; 5,110,587;5,112,749; 5,174,993; 5,223,424; 5,225,336; 5,240,703; 5,242,829;5,294,441; 5,294,548; 5,310,668; 5,387,744; 5,389,368; 5,424,065;5,451,499; 5,453,364; 5,462,734; 5,470,734; 5,474,935; 5,482,713;5,591,439; 5,643,579; 5,650,309; 5,698,202; 5,955,088; 6,034,298;6,042,836; 6,156,319 and 6,589,529, which are each incorporated hereinby reference.

The vaccine provided may be used to induce immune responses includingtherapeutic or prophylactic immune responses. Antibodies and/or killer Tcells may be generated which are directed to the consensus prostateantigen. Such antibodies and cells may be isolated.

The vaccine can further comprise a pharmaceutically acceptableexcipient. The pharmaceutically acceptable excipient can be functionalmolecules as vehicles, adjuvants, carriers, or diluents. Thepharmaceutically acceptable excipient can be a transfection facilitatingagent, which can include surface active agents, such asimmune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPSanalog including monophosphoryl lipid A, muramyl peptides, quinoneanalogs, vesicles such as squalene and squalene, hyaluronic acid,lipids, liposomes, calcium ions, viral proteins, polyanions,polycations, or nanoparticles, or other known transfection facilitatingagents.

The transfection facilitating agent is a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. The transfectionfacilitating agent is poly-L-glutamate, and more preferably, thepoly-L-glutamate is present in the vaccine at a concentration less than6 mg/ml. The transfection facilitating agent can also include surfaceactive agents such as immune-stimulating complexes (ISCOMS), Freundsincomplete adjuvant, LPS analog including monophosphoryl lipid A,muramyl peptides, quinone analogs and vesicles such as squalene andsqualene, and hyaluronic acid can also be used administered inconjunction with the genetic construct. In some embodiments, the DNAvector vaccines can also include a transfection facilitating agent suchas lipids, liposomes, including lecithin liposomes or other liposomesknown in the art, as a DNA-liposome mixture (see for example WO9324640),calcium ions, viral proteins, polyanions, polycations, or nanoparticles,or other known transfection facilitating agents. Preferably, thetransfection facilitating agent is a polyanion, polycation, includingpoly-L-glutamate (LGS), or lipid. Concentration of the transfectionagent in the vaccine is less than 4 mg/ml, less than 2 mg/ml, less than1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml, less than 0.250mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml, or less than 0.010mg/ml.

The pharmaceutically acceptable excipient may be an adjuvant. Theadjuvant may be other genes that are expressed in alternative plasmid orare delivered as proteins in combination with the plasmid above in thevaccine. The adjuvant may be selected from the group consisting of:α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, plateletderived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growthfactor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelialthymus-expressed chemokine (TECK), mucosae-associated epithelialchemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 havingthe signal sequence deleted and optionally including the signal peptidefrom IgE. The adjuvant may be IL-12, IL-15, IL-28, CTACK, TECK, plateletderived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growthfactor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or acombination thereof.

Other genes which may be useful adjuvants include those encoding: MCP-1,MIP-1α, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34,GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2,ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40,CD40L, vascular growth factor, fibroblast growth factor, IL-7, nervegrowth factor, vascular endothelial growth factor, Fas, TNF receptor,Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5,KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1,Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1,JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec,TRAILrecDRCS, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND,NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 andfunctional fragments thereof. The vaccine can further comprise a geneticvaccine facilitator agent as described in U.S. Ser. No. 021,579 filedApr. 1, 1994, which is fully incorporated by reference.

5. METHODS OF DELIVERY

Provided herein is a method for delivering the pharmaceuticalformulations, preferably vaccines, for providing genetic constructs andconsensus prostate antigen which comprise epitopes that make themparticular effective immunogens against which an immune response toprostate cancer cells can be induced. The method of delivering thevaccine, or vaccination, can be provided to induce a therapeutic and/orprophylactic immune response. The vaccine can be delivered to anindividual to modulate the activity of the mammal's immune system andenhance the immune response.

Upon delivery of the vaccine to the mammal, and thereupon the vectorinto the cells of the mammal, the transfected cells will express andsecrete the corresponding prostate consensus protein. These secretedproteins, or synthetic antigens, will be recognized by the immunesystem, which will mount an immune response that can include: antibodiesmade against the antigens, and T-cell response specifically against theantigen. In some examples, a mammal vaccinated with the vaccinesdiscussed herein will have a primed immune system.

The vaccine can be delivered to an individual to modulate the activityof the individual's immune system thereby enhancing the immune response.

The vaccine can be delivered in the form of a DNA vaccine and methods ofdelivering a DNA vaccines are described in U.S. Pat. Nos. 4,945,050 and5,036,006, which are both incorporated fully by reference.

The vaccine can be administered to a mammal to elicit an immune responsein a mammal. The mammal can be human, non-human primate, cow, pig,sheep, goat, antelope, bison, water buffalo, bovids, deer, hedgehogs,elephants, llama, alpaca, mice, rats, or chicken, and preferably human,cow, pig, or chicken.

a. Combination Treatments

The pharmaceutical compositions, preferably vaccines, can beadministered in combination with one or more other prostate proteins orgenes. The vaccine can be administered in combination with proteins orgenes encoding adjuvants, which can include: α-interferon (IFN-α),β-interferon (IFN-β), γ-interferon, IL-12, IL-15, IL-28, CTACK, TECK,platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermalgrowth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18,MCP-1, MIP-1α, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin,CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1,ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18,CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7,nerve growth factor, vascular endothelial growth factor, Fas, TNFreceptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF,DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1,Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K,SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec,TRAILrecDRCS, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND,NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, or TAP2, orfunctional fragments thereof.

b. Routes of Administration

The vaccine can be administered by different routes including orally,parenterally, sublingually, transdermally, rectally, transmucosally,topically, via inhalation, via buccal administration, intrapleurally,intravenous, intraarterial, intraperitoneal, subcutaneous,intramuscular, intranasal intrathecal, and intraarticular orcombinations thereof. For veterinary use, the composition can beadministered as a suitably acceptable formulation in accordance withnormal veterinary practice. The veterinarian can readily determine thedosing regimen and route of administration that is most appropriate fora particular animal. The vaccine can be administered by traditionalsyringes, needleless injection devices, “microprojectile bombardmentgone guns”, or other physical methods such as electroporation (“EP”),“hydrodynamic method”, or ultrasound.

The vector of the vaccine can be delivered to the mammal by several wellknown technologies including DNA injection (also referred to as DNAvaccination) with and without in vivo electroporation, liposomemediated, nanoparticle facilitated, recombinant vectors such asrecombinant adenovirus, recombinant adenovirus associated virus andrecombinant vaccinia. The prostate antigen can be delivered via DNAinjection and along with in vivo electroporation.

c. Electroporation

Administration of the vaccine via electroporation of the plasmids of thevaccine may be accomplished using electroporation devices that can beconfigured to deliver to a desired tissue of a mammal a pulse of energyeffective to cause reversible pores to form in cell membranes, and insome embodiments, the pulse of energy is a constant current similar to apreset current input by a user.

In some embodiments where electroporation is utilized, theelectroporation device may comprise an electroporation component and anelectrode assembly or handle assembly. The electroporation component mayinclude and incorporate one or more of the various elements of theelectroporation devices, including: controller, current waveformgenerator, impedance tester, waveform logger, input element, statusreporting element, communication port, memory component, power source,and power switch. The electroporation may be accomplished using an invivo electroporation device, for example CELLECTRA® EP system (InovioPharmaceuticals, Inc., Blue Bell, Pa.) or Elgen electroporator (InovioPharmaceuticals, Inc., Blue Bell, Pa.) to facilitate transfection ofcells by the plasmid.

The electroporation component may function as one element of theelectroporation devices, and the other elements are separate elements(or components) in communication with the electroporation component. Theelectroporation component may function as more than one element of theelectroporation devices, which may be in communication with still otherelements of the electroporation devices separate from theelectroporation component. The elements of the electroporation devicesexisting as parts of one electromechanical or mechanical device may notlimited as the elements can function as one device or as separateelements in communication with one another. The electroporationcomponent may be capable of delivering the pulse of energy that producesthe constant current in the desired tissue, and includes a feedbackmechanism. The electrode assembly may include an electrode array havinga plurality of electrodes in a spatial arrangement, wherein theelectrode assembly receives the pulse of energy from the electroporationcomponent and delivers same to the desired tissue through theelectrodes. At least one of the plurality of electrodes is neutralduring delivery of the pulse of energy and measures impedance in thedesired tissue and communicates the impedance to the electroporationcomponent. The feedback mechanism may receive the measured impedance andcan adjust the pulse of energy delivered by the electroporationcomponent to maintain the constant current.

A plurality of electrodes may deliver the pulse of energy in adecentralized pattern. The plurality of electrodes may deliver the pulseof energy in the decentralized pattern through the control of theelectrodes under a programmed sequence, and the programmed sequence isinput by a user to the electroporation component. The programmedsequence may comprise a plurality of pulses delivered in sequence,wherein each pulse of the plurality of pulses is delivered by at leasttwo active electrodes with one neutral electrode that measuresimpedance, and wherein a subsequent pulse of the plurality of pulses isdelivered by a different one of at least two active electrodes with oneneutral electrode that measures impedance.

The feedback mechanism may be performed by either hardware or software.The feedback mechanism may be performed by an analog closed-loopcircuit. The feedback occurs every 50 μs, 20 μs, 10 μs or 1 μs, but ispreferably a real-time feedback or instantaneous (i.e., substantiallyinstantaneous as determined by available techniques for determiningresponse time). The neutral electrode may measure the impedance in thedesired tissue and communicates the impedance to the feedback mechanism,and the feedback mechanism responds to the impedance and adjusts thepulse of energy to maintain the constant current at a value similar tothe preset current. The feedback mechanism may maintain the constantcurrent continuously and instantaneously during the delivery of thepulse of energy.

Examples of electroporation devices and electroporation methods that mayfacilitate delivery of the DNA vaccines of the present invention,include those described in U.S. Pat. No. 7,245,963 by Draghia-Akli, etal., U.S. Patent Pub. 2005/0052630 submitted by Smith, et al., thecontents of which are hereby incorporated by reference in theirentirety. Other electroporation devices and electroporation methods thatmay be used for facilitating delivery of the DNA vaccines include thoseprovided in co-pending and co-owned U.S. patent application Ser. No.11/874,072, filed Oct. 17, 2007, which claims the benefit under 35 USC119(e) to U.S. Provisional Applications Ser. Nos. 60/852,149, filed Oct.17, 2006, and 60/978,982, filed Oct. 10, 2007, all of which are herebyincorporated in their entirety.

U.S. Pat. No. 7,245,963 by Draghia-Akli, et al. describes modularelectrode systems and their use for facilitating the introduction of abiomolecule into cells of a selected tissue in a body or plant. Themodular electrode systems may comprise a plurality of needle electrodes;a hypodermic needle; an electrical connector that provides a conductivelink from a programmable constant-current pulse controller to theplurality of needle electrodes; and a power source. An operator cangrasp the plurality of needle electrodes that are mounted on a supportstructure and firmly insert them into the selected tissue in a body orplant. The biomolecules are then delivered via the hypodermic needleinto the selected tissue. The programmable constant-current pulsecontroller is activated and constant-current electrical pulse is appliedto the plurality of needle electrodes. The applied constant-currentelectrical pulse facilitates the introduction of the biomolecule intothe cell between the plurality of electrodes. The entire content of U.S.Pat. No. 7,245,963 is hereby incorporated by reference.

U.S. Patent Pub. 2005/0052630 submitted by Smith, et al. describes anelectroporation device which may be used to effectively facilitate theintroduction of a biomolecule into cells of a selected tissue in a bodyor plant. The electroporation device comprises an electro-kinetic device(“EKD device”) whose operation is specified by software or firmware. TheEKD device produces a series of programmable constant-current pulsepatterns between electrodes in an array based on user control and inputof the pulse parameters, and allows the storage and acquisition ofcurrent waveform data. The electroporation device also comprises areplaceable electrode disk having an array of needle electrodes, acentral injection channel for an injection needle, and a removable guidedisk. The entire content of U.S. Patent Pub. 2005/0052630 is herebyincorporated by reference.

The electrode arrays and methods described in U.S. Pat. No. 7,245,963and U.S. Patent Pub. 2005/0052630 may be adapted for deep penetrationinto not only tissues such as muscle, but also other tissues or organs.Because of the configuration of the electrode array, the injectionneedle (to deliver the biomolecule of choice) is also insertedcompletely into the target organ, and the injection is administeredperpendicular to the target issue, in the area that is pre-delineated bythe electrodes The electrodes described in U.S. Pat. No. 7,245,963 andU.S. Patent Pub. 2005/005263 are preferably 20 mm long and 21 gauge.

Additionally, contemplated in some embodiments that incorporateelectroporation devices and uses thereof, there are electroporationdevices that are those described in the following patents: U.S. Pat. No.5,273,525 issued Dec. 28, 1993, U.S. Pat. No. 6,110,161 issued Aug. 29,2000, U.S. Pat. No. 6,261,281 issued Jul. 17, 2001, and U.S. Pat. No.6,958,060 issued Oct. 25, 2005, and U.S. Pat. No. 6,939,862 issued Sep.6, 2005. Furthermore, patents covering subject matter provided in U.S.Pat. No. 6,697,669 issued Feb. 24, 2004, which concerns delivery of DNAusing any of a variety of devices, and U.S. Pat. No. 7,328,064 issuedFeb. 5, 2008, drawn to method of injecting DNA are contemplated herein.The above-patents are incorporated by reference in their entirety.Another embodiment of an electroporation device to be used with thecancer antigens described herein is the Elgen EP device (InovioPharmaceuticals, Inc., Blue Bell, Pa.).

d. Method of Preparing Vaccine

Provided herein is methods for preparing the DNA plasmids that comprisethe DNA vaccines discussed herein. The DNA plasmids, after the finalsubcloning step into the mammalian expression plasmid, can be used toinoculate a cell culture in a large scale fermentation tank, using knownmethods in the art.

The DNA plasmids for use with the EP devices of the present inventioncan be formulated or manufactured using a combination of known devicesand techniques, but preferably they are manufactured using an optimizedplasmid manufacturing technique that is described in a licensed,co-pending U.S. provisional application U.S. Ser. No. 60/939,792, whichwas filed on May 23, 2007. In some examples, the DNA plasmids used inthese studies can be formulated at concentrations greater than or equalto 10 mg/mL. The manufacturing techniques also include or incorporatevarious devices and protocols that are commonly known to those ofordinary skill in the art, in addition to those described in U.S. Ser.No. 60/939,792, including those described in a licensed patent, U.S.Pat. No. 7,238,522, which issued on Jul. 3, 2007. The above-referencedapplication and patent, U.S. Ser. No. 60/939,792 and U.S. Pat. No.7,238,522, respectively, are hereby incorporated in their entirety.

EXAMPLES

The present invention is further illustrated in the following Examples.It should be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, various modifications of the invention in addition tothose shown and described herein will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Example 1

Consensus immunogens for PSA and PSMA were designed from the availablefull-length human and macaque sequences in the GenBank database aspreviously described in Laddy, D. J., Yan, J., Corbitt, N., Kobasa, D.,Kobinger, G. P., Weiner, D. B. (2007). Immunogenicity of novelconsensus-based DNA vaccines against avian influenza. Vaccine. 25,2984-2989, and Laddy, D. J., Yan, J., Kutzler, M., Kobasa, D., Kobinger,G. P., Khan, A. S., Greenhouse, J., Sardesai, N. Y., Draghia-Akli, R.,Weiner, D. B. (2008). Heterosubtypic Protection against Pathogenic Humanand Avian Influenza Viruses via In Vivo Electroporation of SyntheticConsensus DNA Antigens. PLoS ONE. 3,e2517.

The consensus antigen sequences were synthesized by GeneScript(Piscataway, N.J.). An HA tag was included in the C-terminus of theantigen sequence. The antigen sequences were optimized for mRNAstability and codon usage in humans. The final sequences were cloned inthe BamHI and XhoI sites of the pVAX1 vector (Invitrogen, Carlsbad,Calif.).

A consensus PSA antigen 1 (SEQ ID NO:2) was generated. This sequence,which comprises 261 amino acids, was compared to each of the PSAsequences set forth in Table 1. The PSA sequences used include two humansequences, a sequence from M. fascicularis, and a sequence from M.mulatta. Table 1 includes the SEQ ID NO: and Accession number for eachsequence used in the comparison with consensus PSA antigen 1 (SEQ IDNO:2).

TABLE 1 SEQ Number % homology ID Species and Accession of amino to SEQID NO protein Number acids NO: 2 17 H. sapiens PSA NP001639.1 261 91isol 18 H. sapiens PSA gbAAA60193.1 262 91 19 M. fascicularis Q6DT45.1261 95 KLK3 20 M. mulatta PSA NP001036241.1 p 261 96

A multiple sequence alignment of H. Sapiens (SEQ ID NO:17 and SEQ IDNO:18), M. mulatta (SEQ ID NO:20) and M. facicularis (SEQ ID NO:19) PSAsequences was generated with the consensus PSA antigen 1 (SEQ ID NO:2).KLK3 (kallikrein 3) is the gene encoding PSA and is pseudonymous withPSA. The PSA antigen 1 is 91% homologous to H. sapiens, 96% homologousto M. mulatta and 95% homologous M. facicularis full-length PSA proteinsequences.

Example 2

A consensus PSA antigen 2 (SEQ ID NO:4) was generated. This sequence,which comprises 279 amino acids including an IgE leader sequence, wascompared to each of the PSA sequences set forth in Table 2. The PSAsequences used include two human sequences, a sequence from M.fascicularis, and a sequence from M. mulatta. Table 2 includes the SEQID NO: and Accession number for each sequence used in the comparisonwith consensus PSA antigen 2 (SEQ ID NO:4).

TABLE 2 Number % homology SEQ Species and Accession of amino to SEQ IDID NO protein Number acids NO: 4 17 H. sapiens PSA NP001639.1 261 91isol 18 H. sapiens PSA gbAAA60193.1 262 90 19 M. fascicularis Q6DT45.1261 95 KLK3 21 M. mulatta PSA AAZ82258.1 244 95

A multiple sequence alignment of H. Sapiens (SEQ ID NO:17 and SEQ IDNO:18), M. mulatta (SEQ ID NO:21) and M. facicularis (SEQ ID NO:19) PSAsequences was generated with the consensus PSA antigen 1 (SEQ ID NO:4).KLK3 (kallikrein 3) is the gene encoding PSA and is pseudonymous withPSA. The PSA antigen 1 is 90-91% homologous to H. sapiens and 95%homologous M. facicularis full-length PSA protein sequences, and 95%homologous to M. mulatta partial PSA protein sequence.

Example 3

A consensus PSMA antigen 1 (SEQ ID NO:6) was generated. This sequence,which comprises 750 amino acids was compared to each of the PSMAsequences set forth in Table 3. The PSMA sequences used include twohuman sequences and a sequence from M. mulatta. Table 3 includes the SEQID NO: and Accession number for each sequence used in the comparisonwith consensus PSMA antigen 1 (SEQ ID NO:6).

TABLE 3 SEQ Number % homology ID Species and Accession of amino to SEQID NO protein Number acids NO: 6 22 H. sapiens PSMA NP_004467.1 750 96GCPII_isol 23 H. sapiens PSMA AAC83972.1 749 96 24 M. mulatta GCPIIXP_001096141.2 735 94 isol

A multiple sequence alignment of H. Sapiens and M. mulatta PSMAsequences was generated with PSMA antigen 1. The PSMA antigen 1consensus sequence (SEQ ID NO:6) is 96% homologous to H. sapiens PSMA(SEQ ID NO:22 and SEQ ID NO:23) and 94% homologous to M. mulattafull-length PSMA protein sequence (SEQ ID NO:24).

Example 4

A consensus PSMA antigen 2 (SEQ ID NO:8) was generated. This sequence,which comprises 766 amino acids including an IgE leader sequence, wascompared to each of the PSMA sequences set forth in Table 4. The PSMAsequences used include two human sequences and a sequence from M.mulatta. Table 4 includes the SEQ ID NO: and Accession number for eachsequence used in the comparison with consensus PSMA antigen 2 (SEQ IDNO:8).

TABLE 4 SEQ Number % homology ID Species and Accession of amino to SEQID NO protein Number acids NO: 8 22 H. sapiens PSMA NP_004467.1 750 96GCPII_isol 23 H. sapiens PSMA AAC83972.1 749 96 24 M. mulatta GCPIIXP_001096141.2 735 94 isol 25 M. mulatta GCPII XP_002799784.1 704 94iso2

A multiple sequence alignment of H. sapiens (SEQ ID NO:22 and SEQ IDNO:23) and M. mulatta PSMA sequences (SEQ ID NO:24 and SEQ ID NO:25) wasgenerated with PSMA antigen 2. The PSMA antigen 2 consensus sequence(SEQ ID NO:8) is 96% homologous to H. sapiens PSMA protein sequences and94% homologous to M. mulatta PSMA protein sequences.

Example 5

A consensus STEAP antigen 1 (SEQ ID NO:10) was generated. This sequence,which comprises 339 amino acids was compared to each of the STEAPsequences set forth in Table 5. The STEAP sequences used include twofull length human sequences, a full length sequence from M. mulatta andtwo shorter human sequences. Table 5 includes the SEQ ID NO: andAccession number for each sequence used in the comparison with consensusSTEAP antigen 1 (SEQ ID NO:10).

TABLE 5 SEQ Number % homology ID Species and Accession of amino to SEQID NO protein Number acids NO: 10 26 H. sapiens STEAP1 NP_036581.1 33999 27 H. sapiens STEAP1 Gb_EAL24167.1 339 99 28 M. mulatta STEAP1XP_001103605.1 339 98 29 H. sapiens STEAP1 EAW93751.1 259 94 CRA b 30 H.sapiens STEAP1 EAW93749.1 258 94 isofor

A multiple sequence alignment of H. Sapiens and M. mulatta STEAPsequences was generated with the consensus STEAP antigen 1. The STEAPantigen 1 consensus sequence (SEQ ID NO:10) is 99% homologous to humanfull-length isoforms (SEQ ID NO:26 and SEQ ID NO:27), 94% homologous toshorter H. sapiens isoforms (SEQ ID NO:29 and SEQ ID NO:30), and 94%homologous to M. mulatta full-length STEAP1 protein sequence (SEQ IDNO:28).

Example 6

A consensus STEAP antigen 2 (SEQ ID NO:12) was generated. This sequence,which comprises 356 amino acids was compared to each of the STEAPsequences set forth in Table 6. The STEAP sequences used include twofull length human sequences, a full length sequence from M. mulatta andtwo shorter human sequences. Table 6 includes the SEQ ID NO: andAccession number for each sequence used in the comparison with consensusSTEAP antigen 2 (SEQ ID NO:12).

TABLE 6 SEQ Number % homology ID Species and Accession of amino to SEQID NO protein Number acids NO: 12 26 H. sapiens STEAP1 NP_036581.1 33994 27 H. sapiens STEAP1 Gb_EAL24167.1 339 94 28 M. mulatta STEAP1XP_001103605.1 339 94 29 H. sapiens STEAP1 EAW93751.1 259 88 CRA b 30 H.sapiens STEAP1 EAW93749.1 258 88 isofor

A multiple sequence alignment of H. Sapiens and M. mulatta STEAP1sequences was generated with the consensus STEAP1 antigen 2. The STEAP1antigen 2 consensus sequence (SEQ ID NO:12) is 94% homologous tofull-length human isoforms (SEQ ID NO:26 and SEQ ID NO:27), 88%homologous to shorter H. sapiens isoforms (SEQ ID NO:29 and SEQ IDNO:30), and 94% homologous to M. mulatta full-length STEAP1 proteinsequences (SEQ ID NO:28).

Example 7

A consensus PSCA antigen (SEQ ID NO:14) was generated. This sequence,which comprises 131 amino acids included the IgE leader sequence wascompared to PSCA sequence set forth in Table 7. The PSCA sequence usedwas a full length human sequence. Table 7 includes the SEQ ID NO: andAccession number for the sequence used in the comparison with consensusPSCA antigen (SEQ ID NO:14).

TABLE 7 Number % homology SEQ Species and Accession of amino to SEQ IDID NO protein Number acids NO: 14 31 H. sapiens PSCA NP_005663.2 114 87

A multiple sequence alignment of H. Sapiens PSCA sequence (SEQ ID NO:31)was generated with the consensus PSCA antigen (SEQ ID NO:14). The PSCAantigen consensus sequence is 87% homologous to full-length H. sapiensPSCA.

Example 8

In vitro translation performed to confirm the expression of the PSA andPSMA antigens. The TNT® Quick Coupled Transcription/Translation Systemand 35S-methionine (Promega) were used. The pVAX vector alone (negativecontrol) or pVAX backbone with the PSA or PSMA antigen inserts and35S-methionine was added to the reaction mixture according to themanufacturer's instructions. The reaction was carried out at 30° C. for2 hours. Labeled proteins were immunoprecipitated with anti-HA AffinityGel (Sigma, St. Louis, Mo.) by rotation overnight inradioimmunoprecipitation assay (RIPA) buffer at 4° C. Theimmunoprecipitated proteins were electrophoresed on a SDS-PAGE gel thatwas subsequently fixed and dried. Expression of the 35S-labeled proteinswas detected by autoradiography. The results are shown in FIG. 1.

Example 9

Cellular immunogenicity of the PSA and PSMA antigens was determined byInterferon-gamma ELISpot.

Female 4 to 6-week-old BALB/c mice were purchased from JacksonLaboratories (Bar Harbor, Me.). All animals were housed in atemperature-controlled, light-cycled facility at the University ofPennsylvania. Animal care was carried out according to the guidelines ofthe National Institutes of Health and the University of PennsylvaniaInstitutional Care and Use Committee.

For cellular immunogenicity studies, 10 or 20 mg of each antigen wasdelivered to the tibialis anterior muscle of Balb/c mice byintramuscular injection followed by electroporation using the CELLECTRA®adaptive constant current device (Inovio Pharmaceuticals, Inc., BlueBell, Pa.). Mice (n=5 per group) received 2 immunizations at weeks 0 and2. Two 0.1 Amp constant current square-wave pulses were deliveredthrough a triangular 3-electrode array consisting of 26-gauge solidstainless steel electrodes. Each pulse was 52 milliseconds in lengthwith a 1 second delay between pulses. The mice received a total of 2immunizations that were administered 2 weeks apart. Mice were humanelysacrificed 1 week after the second immunization for analysis of cellularand humoral immune responses.

Cellular and responses were assessed 1 week after the last immunization(week 5). ELISpot analysis was used to determine antigen-specificsecretion of IFNγ. Mouse IFNγ capture antibody (R&D Systems,Minneapolis, Minn.) was used to coat flat-bottom Immobilon-P plates(Millipore, Billerica, Mass.) overnight at 4° C. Splenocytes wereaseptically isolated and resuspended at in R10 media (Rosewell ParkMemorial Institute medium 1640 with supplemented with 10% fetal bovineserum, 1% antibiotic-antimycotic and 0.1% 2-mercaptoethanol). 2×10⁵splenocytes from immunized mice were added in to each well of the96-well plate and stimulated overnight at 37° C., 5% CO2, in thepresence of R10 (negative control), concanavalin A (positive control)(Sigma, St. Louis, Mo.) or antigen-specific peptide pools. The next day,mouse IFNγ detection antibody (R&D Systems, Minneapolis, Minn.) wasadded to the plates that were then incubated overnight at 4° C. Thefollowing day, streptavidin-ALP (MabTech, Sweden) was added to theplates for 2 hours and antigen-specific spots were visualized withBCIP/NPT substrate (MabTech, Sweden). PSA and PSMA peptides were 15-merpeptides spanning the entire length of the consensus immunogen, notincluding the HA tag or leader sequence, overlapping by 11 amino acids,and were synthesized by GenScript (Piscataway, N.J.). PSA and PSMApeptides were used at a final concentration of 1.0 μg/mL for eachpeptide. IFNγ ELISpot was used to evaluate antigen-specific cellularresponses 1 week after the last immunization. For PSA, IFNγ responseswere similar for the 10 μg (772.2+/−138.2 SFU) and 20 μg (771.1+/−155.2SFU) vaccine doses (FIG. 2A). In contrast, there was a dose-dependantincrease in PSMA-specific FNγ responses with 20 microgram of the vaccine(1585.0+/−194.0 SFU) as compared to 10 μg of the vaccine (1047.2+/−160.7SFU) (FIG. 2B). Minimal background was observed for PSA or PSMAresponses in naïve mice.

Example 10 Vaccine-Induced CD4+ and CD8+ T Cell Production of IFNγ, IL-2and TNFα

Cellular immunogenicity was further characterized by flow cytometry forthe co-delivery of the PSA and PSMA vaccines. Antigen-specific CD4+ andCD8+ T cell production of IFNγ, IL-2 and TNFα was determined for thetotal vaccine-specific response and the PSA and PSMA components of thetotal vaccine-specific response (n=5).

Cellular immune responses were also determined by intracellular cytokinestaining and flow cytometry using the CytoFix/CytoPerm kit permanufacteurer's instructions (BD Biosciences, San Diego, Calif.).Splenocytes harvested from immunized mice were washed with PBS and thenresuspended in R10 media to a final concentration of 10⁷ cells/ml. Cellswere seeded in 96-well round bottom plates in a volume of 100 μl and anadditional 100 μl of R10 media (negative control), media containingantigen-specific peptides pools or media containing phorbol myristateacetate (PMA, 10 ng/ml) and ionomycin (250 ng/ml; positive control)(Sigma, St. Louis, Mo.) was added and plates were incubated at 37° C.,5% CO2, for 6 hours. All stimulation media contained 1 μg/μL each ofGolgiPlug and GolgiStop (BD Biosciences, San Diego, Calif.). At the endof the incubation period plates were spun down and washed twice withPBS. Cells were then stained with a violet dye for viability (LIVE/DEADViolet Viability Dye, Invitrogen; Carlsbad, Calif.) for 30 minutes at 4°C. After washing as above with PBS, cells were stained externally for 30minutes with anti-CD4 PerCPCy5.5 and anti-CD8 APC at 4° C., followed byfixing and permeabilization. Anti-CD3 PE-Cy5, anti-IL-2 PE, anti-IFNγAlexaFluor-700 and anti-TNFα FITC (BD Biosciences, San Diego, Calif.)were added and cells were incubated again at 4° C. for 30 minutes. Cellswere given a final wash with PBS and fixed in 1% PFA.

Co-delivery of the PSA and PSMA vaccine induced robust CD4+ secretion ofIFNγ, IL-2 and TNFα. The percentage of PSA-specific (0.21%) andPSMA-specific (0.24%) IFNγ producing CD4+ T cells contributed equally tothe total vaccine-specific CD4+ T cell IFNγ response (0.44%) (FIG. 3A).PSMA-specific CD4+ T cells producing IL-2 (1.08%) comprised the majorityof the total percentage of CD4+ T cells producing vaccine-specific IL-2(1.40%) (FIG. 3B). The percentage of PSA (0.31%) and PSMA (0.29%)induced CD4+ T cell production of TNFα contributed equally to the totalvaccine-specific response (0.60%) (FIG. 3C). Overall, CD4+ T cellresponses were well balanced between PSA and PSMA, with the exception ofPSMA inducing the majority of the vaccine-specific CD4+ T cell IL-2production.

The vaccine induced strong antigen-specific CD8+ T cell production ofIFNγ and IL-2 and, to a lesser extent, TNFα. Both PSA (0.70%) and PSMA(0.67%) induced robust CD8+ T cell IFNγ production. In fact,vaccine-specific CD8+ T cells secreting of IFNγ comprised 1.37% of thetotal CD8+ T cell population (FIG. 4A). The vaccine also induced astrong CD8+ T cell IL-2 response (1.54%). Similar to the CD4+ T cellIL-2 response, the percentage of PSMA-specific (1.06%) CD8+ T cellssecreting IL-2 was approximately 2-fold higher than PSA-specific (0.47%)(FIG. 4B). The total percentage of vaccine-specific CD8+ T cellproduction of TNFα (0.11%) was in response to the PSA component of thevaccine (FIG. 4C). In summary, there was a high percentage ofvaccine-specific CD8+ T cells production of IFNγ and IL-2. Similar toCD4+ T cell responses, IFNγ production was equally balanced between PSAand PSMA and the magnitude of the IL-2 PSMA-specific response wasgreater than that of the PSA-specific response.

Example 11 PSA-Specific IgG Seroconversion

Antibody response can play an important role in tumor immunotherapy.Accordingly we next examined this parameter of the immune response tothe PSA antigen based on protein target availability.

To determine PSA-specific sera antibody titers, 96-well Nunc-ImmunoMaxiSorp plates (Nunc, Rochester, N.Y.) were coated overnight at 4° C.with 1 μg/well of recombinant PSA protein (Fitzgerald Industries, Acton,Mass.) diluted in PBS. Plates were washed with PBS, 0.05% Tween 20(PBST), blocked for 1 hour at room temperature with 10% BSA/PBST, andincubated with serial dilutions of serum from immunized or naïve animalsfor 1 hour at room temperature. Plates were then washed 3 times withPBST and goat anti-mouse IgG (Santa Cruz, Santa Cruz, Calif.) was addeda dilution of 1:5,000 in PBST. Bound enzyme was detected by SigmaFASTO-phenylenediamine dihydrochloride (OPD; Sigma-Aldrich, St. Louis, Mo.),and the optical density was determined at 450 nm on a Biotek (Winooski,Vt.) plate reader as shown in FIG. 5B. Endpoint titers were determinedas previously described (Frey, A. et al. 1998). Briefly, the upperprediction limit was calculated using the Student t-distribution. Themathematical formula that defines the upper prediction limit isexpressed as the standard deviation multiplied by a factor that wasbased on the number of negative controls (n=5) and the confidence level(95%). The endpoint titer was reported as the reciprocal of the lastdilution above the upper predication limit.

In addition to conferring robust cellular-mediated immunity, the PSAvaccine also induced strong antigen-specific humoral responses. Antibodytiters were determined by ELISA in sera isolated from mice one weekafter the last immunization (n=5). The vaccine induced an averagePSA-specific antibody endpoint titer of 4,427 (range 1581-15,811) (FIG.5A). The longevity of these responses may be important as well.

Example 12

Prostate specific antigen amino acid sequences available on GenBankinclude the following: gb_EAW71923.1_(—) H.sapiens_klk3_CRAb;_(—)001639.1_(—) H.sapiens_PSA_iso1_preproprotein; gb_AAA59995.1_(—)H.sapiens_PSA_precursor; gb_AAA60193.1_(—) H.sapiens_PSA;gb_EAW71933.1_(—) H.sapiens_klk3_CRA_(—)1; NP_(—)001025218.1_(—)H.sapiens_PSA_iso3_preproprotein; gb_CAD54617.1_(—) H.sapiens_PSA;gb_CAD30844.1_(—) H.sapiens_PSA; gb_AAA59996.1_(—)H.sapiens_PSA_precursor; gb_AAD14185.1_(—) H.sapiens_PSA; Q6DT45.1_(—)M.fascicularis_KLK3; NP_(—)001036241.1_(—) M.mulatta_PSA_precursor;AAZ82258.1_(—) M.mulatta_PSA; AAZ82255.1_(—) G.gorilla_PSA;gi|1163838666|ref|NP_(—)001106216.1|plasma kallikrein [Papio anubis];gi|73746696|gb|AAZ82261.1|prostate specific antigen [Papio anubis];i|73746692|gb|AAZ82259.1|prostate specific antigen [Erythrocebus patas];gi|73746694|gb|AAZ82260.1|prostate specific antigen [Cercopithecuscephus]; gi|73746682|gb|AAZ82254.1|prostate specific antigen [Panpaniscus]; gi|73746680|gb|AAZ82253.1|prostate specific antigen [Pantroglodytes]; gi|73746686|gb|AAZ82256.1|prostate specific antigen [Pongopygmaeus]; and 3746688|gb|AAZ82257.1|prostate specific antigen [Nomascusgabriellae].

PSMA amino acid sequences available on GenBank include the following:NP_(—)004467.1_Human_GCPII_iso1; Human PSMA_AAC83972.1;M.mulatta_GCPII_iso1 XP_(—)001096141.2; and M.mulatta_GCPII_iso2XP_(—)002799784.1.

STEAP amino acid sequences available on GenBank include the following:NP036581.1_Human_STEAP1; EAL24167.1_Human_STEAP1;XP001103605.1M.mulatta_STEAP1_iso3; EAW93751.1_HumanSTEAP1_CRAb;EAW93749.1_Human_STEAP1_CRAa; XP001164838.1_(—) P.troglodytes_STEAPiso2;XP002818311.1_(—) P.abelii_STEAP1; NP001162459.1_(—) P.anubis_STEAP1;NP_(—)999470.1_(—) S.scrofa_STEAP1; and NP_(—)081675.2_(—)M.musculusSTEAP1.

NP_(—)005663.2_Human_PSCA is the accession number of a PSCA amino acidsequence available on GenBank.

1. A nucleic acid molecule comprising a coding sequence encoding one ormore proteins selected from the group comprising: a) SEQ ID NO:2, aprotein that is 98% homologous to SEQ ID NO:2, provided amino acids 69,78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 of SEQID NO:2 are conserved, or an immunogenic fragment of SEQ ID NO:2comprising amino acids corresponding to at least 256 amino acid residuesof SEQ ID NO:2 provided amino acids 69, 78, 80, 82, 102, 110, 137, 139,165, 189, 203, 220, 232 and 248 of SEQ ID NO:2 are conserved; b) SEQ IDNO:4, a protein that is 98% homologous to SEQ ID NO:4, provided aminoacids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249,255, 265, 271 or 275 of SEQ ID NO:4 are conserved, or an immunogenicfragment of SEQ ID NO:4 comprising amino acids corresponding to at least274 amino acid residues of SEQ ID NO:4, provided amino acids 21, 86,127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 or275 of SEQ ID NO:4 are conserved; c) SEQ ID NO:6, a protein that is 98%homologous to SEQ ID NO:6, provided amino acids 14, 15, 32, 47, 58, 79,111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and734 of SEQ ID NO:6 are conserved, or an immunogenic fragment of SEQ IDNO:6 comprising amino acids corresponding to at least 735 amino acidresidues of SEQ ID NO:6, provided amino acids 14, 15, 32, 47, 58, 79,111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and734 of SEQ ID NO:6 are conserved; d) SEQ ID NO:8, a protein that is 98%homologous to SEQ ID NO:8, provided amino acids 20, 30, 31, 48, 63, 74,95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676,679, 749 and 750 of SEQ ID NO:8 are conserved, or an immunogenicfragment of SEQ ID NO:8 comprising amino acids corresponding to at least751 amino acid residues of SEQ ID NO:8, provided amino acids 20, 30, 31,48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640,669, 676, 679, 749 and 750 of SEQ ID NO:8 are conserved; e) SEQ IDNO:10, a protein that is 98% homologous to SEQ ID NO:10, provided, or animmunogenic fragment of SEQ ID NO:10 comprising amino acidscorresponding to at least 333 amino acid residues of SEQ ID NO:10; f)SEQ ID NO:12, a protein that is 98% homologous to SEQ ID NO:12, or animmunogenic fragment of SEQ ID NO:12 comprising amino acidscorresponding to at least 349 amino acid residues of SEQ ID NO:12; or g)SEQ ID NO:14, a protein that is 98% homologous to SEQ ID NO:14, or animmunogenic fragment of SEQ ID NO:14 comprising amino acidscorresponding to at least 129 amino acid residues of SEQ ID NO:14. 2.The nucleic acid molecule of claim 1 encoding one or more proteinsselected from the group comprising: elements a), b), c), or d).
 3. Thenucleic acid molecule of claim 1 encoding one or more proteins selectedfrom the group comprising: at least one selected from either elements a)or b), and at least one selected from either elements c) or d).
 4. Thenucleic acid molecule of claim 1 encoding one or more proteins selectedfrom the group comprising: SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ IDNO:8; SEQ ID NO:10; SEQ ID NO:12; or SEQ ID NO:14.
 5. The nucleic acidmolecule of claim 1 encoding one or more proteins selected from thegroup comprising: SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; or SEQ ID NO:8.6. The nucleic acid molecule of claim comprising one or more sequencesselected from the group comprising: a) SEQ ID NO:1, or a coding sequencethat is 98% homologous to SEQ ID NO:1; b) SEQ ID NO:3, or a codingsequence that is 98% homologous to SEQ ID NO:3; c) nucleotides 1-2250 ofSEQ ID NO:5, or a coding sequence that is 98% homologous to nucleotides1-2250 of SEQ ID NO:5; d) nucleotides 1-2301 of SEQ ID NO:7, or a codingsequence that is 98% homologous to nucleotides 1-2301 of SEQ ID NO:7; e)SEQ ID NO:9, or a coding sequence that is 98% homologous to SEQ ID NO:9;f) SEQ ID NO:11, or a coding sequence that is 98% homologous to SEQ IDNO:11; or g) SEQ ID NO:13, or a coding sequence that is 98% homologousto SEQ ID NO:13.
 7. The nucleic acid molecule of claim 6 comprising oneor more nucleotide sequences selected from the group comprising:elements a), b), c), or d).
 8. The nucleic acid molecule of claim 6comprising one or more nucleotide sequences selected from the groupcomprising: at least one selected from either elements a) or b), and atleast one selected from either elements c) or d).
 9. The nucleic acidmolecule of claim 6 comprising one or more nucleotide sequences selectedfrom the group comprising: SEQ ID NO:1; SEQ ID NO:3; nucleotides 1-2250of SEQ ID NO:5; nucleotides 1-2301 of SEQ ID NO:7; SEQ ID NO:9; SEQ IDNO:11; or SEQ ID NO:13.
 10. The nucleic acid molecule of claim 1 whereinthe nucleic acid molecule is a plasmid.
 11. The nucleic acid molecule ofclaim 1 wherein the nucleic acid molecule is an expression vector andsequences encoding said one more proteins are operable linked toregulatory elements.
 12. A method of treating an individual who has beendiagnosed with prostate cancer comprising administering a nucleic acidmolecule of claim 1 to an individual.
 13. A protein selected from thegroup consisting of: a) SEQ ID NO:2, a protein that is 98% homologous toSEQ ID NO:2, provided amino acids 69, 78, 80, 82, 102, 110, 137, 139,165, 189, 203, 220, 232 and 248 of SEQ ID NO:2 are conserved; or animmunogenic fragment of SEQ ID NO:2 comprising amino acids correspondingto at least 256 amino acid residues of SEQ ID NO:2, provided amino acids69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 ofSEQ ID NO:2 are conserved; b) SEQ ID NO:4, a protein that is 98%homologous to SEQ ID NO:4, provided amino acids 21, 86, 127, 129, 154,156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 or 275 of SEQ IDNO:4 are conserved; or an immunogenic fragment of SEQ ID NO:4 comprisingamino acids corresponding to at least 274 amino acid residues of SEQ IDNO:4, provided amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206,218, 220, 237, 249, 255, 265, 271 or 275 of SEQ ID NO:4 are conserved;c) SEQ ID NO:6; a protein that is 98% homologous to SEQ ID NO:6,provided amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350,475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO:6 areconserved; or an immunogenic fragment of SEQ ID NO:6 comprising aminoacids corresponding to at least 735 amino acid residues of SEQ ID NO:6,provided amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350,475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO:6 areconserved; d) SEQ ID NO:8, a protein that is 98% homologous to SEQ IDNO:8, provided amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239,336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 ofSEQ ID NO:8 are conserved, or an immunogenic fragment of SEQ ID NO:8comprising amino acids corresponding to at least 751 amino acid residuesof SEQ ID NO:8, provided amino acids 20, 30, 31, 48, 63, 74, 95, 127,173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and750 of SEQ ID NO:8 are conserved; e) SEQ ID NO:10, a protein that is 98%homologous to SEQ ID NO:10; or an immunogenic fragment of SEQ ID NO:10comprises amino acids corresponding to at least 333 amino acid residuesof SEQ ID NO:10; f) SEQ ID NO:12, a protein that is 98% homologous toSEQ ID NO:12, or an immunogenic fragment of SEQ ID NO:12 comprises aminoacids corresponding to at least 349 amino acid residues of SEQ ID NO:12;g) SEQ ID NO:14, a protein that is 98% homologous to SEQ ID NO:14, or animmunogenic fragment of SEQ ID NO:14 comprising amino acidscorresponding to at least 129 amino acid residues of SEQ ID NO:14; or h)a signal peptide linked to amino acids 19-131 of SEQ ID NO:14, a proteinthat has a signal peptide linked to an amino acid sequence that is 98%homologous to amino acids 19-131 of SEQ ID NO:14, or a protein that hasa signal peptide linked to an immunogenic fragment of amino acids 19-131of SEQ ID NO:14, the fragment comprising at least 110 amino acidresidues of SEQ ID NO:14 and linked to a signal peptide.
 14. The proteinof claim 13 encoding one or more proteins selected from the groupcomprising: elements a), b), c), or d).
 15. The protein of claim 13encoding one or more proteins selected from the group comprising: atleast one selected from either elements a) or b), and at least oneselected from either elements c) or d).
 16. The protein of claim 13encoding a protein selected from the group comprising: SEQ ID NO:2; SEQID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; or SEQ IDNO:14.
 17. The protein of claim 13 encoding a protein selected from thegroup comprising: SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; or SEQ ID NO:8.18. A method of treating an individual who has been diagnosed withprostate cancer comprising delivering to said individual a protein ofclaim
 13. 19. A pharmaceutical composition comprising the nucleic acidmolecule of claim 1 and a pharmaceutically acceptable excipient.